© 2012 Nature America, Inc. All rights reserved. method was developed about a century ago by Spalteholz, who who Spalteholz, by ago century a about developed was method the different throughout tissue layers. For this unobstructed purpose, the first tissue clearing travels and scatter not does light ing tissues thick imaging for method prominent a become has solvent, the to layers tissue different of indexes refractory the matching by transparent tissue biological in a lower resolution and depth imaging efficiency,resulting emission and excitation its loses and scatters structures different through traveling light imaging The indices. various cellular and extracellular contain which structures tissues, with thick different the refractivethrough light imaging of tering scat of because mostly limited is tissues thick of imaging optical be imaged at high spatial resolution sectioning. without However, are large observed within organs. cells target of fractions only small when studies immunology and axonal problems Similar couldfragments. canceroccur in of tion regions of the regenerating axons cannot be identified by visualiza axons spared be might be interpreted as regenerated axons even though they could profiles axonal fragmented these confusion, this of Because lost. is structures axonal of information 3D the hence, section; each the microscope, only short fragments of axons are observed within is sectioned. Subsequently, when tissue sections are analyzed under cord,nerve) spinal optic (e.g., interest of tissue the that common axonin injured studies, axons,the regeneration after labeling is it example,Fortissues. within cells of distribution the and ponents com structural of picture 3D a obtaining for limitations major of blocks of frozen or fixed tissue for microscopy. This process has sections thin make to necessary is study, it histology typical a In I offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies. tissues. for performing 3DI which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DI subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We and they present major limitations for 3D tissue reconstruction. organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, T Published online 11 October 2012; doi:10.1038/nprot.2012.119 Bonn, Germany. Correspondence should be addressed to A.E. ( Immunology, Genentech, South San Francisco, California, USA. University Oldenburg, of Oldenburg, Germany. Technology,of Vienna, Austria. 1 Farida Hellal Ali Ertürk organs using 3DISCO Three-dimensional imaging of solvent-cleared organs such as the heart used a mixture of benzyl alcohol and methyl salicylate to clear large Department of Neuroscience,of Department Genentech, South San Francisco, California, USA. NTRO he he examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of Optical clearing of tissues by organic solvents, which make the the make solvents, which organic by tissues of clearing Optical should ideally problems,tissues and To limitations avoidthese D UCT T he he tissue clearing takes as little as 3 h, and imaging can be completed in ~45 min. 3DI 1 I , Klaus Becker ON 7 , Frank Bradke 1 . In addition, the growth trajectories and target target and trajectories growth the addition, In . SCO and present its application to various microscopy techniques, including example results from various mouse 5, 6 . In general, the first step of tissue clearing 3 Center for Brain Research, Medical University of Vienna, Bioelectronics, of Section Vienna, Austria. 2 , 3 , Nina Jährling 7 2, , Morgan Sheng 4 . In cleared tissues, the imag the tissues, cleared In . 5 Max Planck Institute Psychiatry,of Neuronal ResearchPlasticity Group, Munich, Germany. 2, 3 . 2 – [email protected] 4 7 , Christoph P Mauch Deutsches Deutsches Zentrum für neurodegenerative (DZNE),Erkrankungen Axonal Growth and Regeneration, 1 & Hans-Ulrich Dodt 2 - - - - Department of Bioelectronics, of Institute Department Solid State of Electronics, Vienna University T ). he he introduction of methods to achieve the optical clearing and had limited success on the adult brain. Recently, we developed a developed Recently, we brain. adult the on success limited had signal fluorescent its with BABB, could both fully clear the adult spinal cord and preserve tetrahydrofuran (THF)—instead of alcohol—use, in combination Therefore, we screened for new clearing chemicals and found that tissue. (CNS) system nervous central adult on work not did thus BABB combination could and in myelinated clear not and tissues fluorescenceexpression strong with tissues fluorescent signal quickly, and hence limited its application as to small such embryonic hippocampi. However, preparations the use smaller of alcohol the degraded for well especially worked which clearing, tissue for BABB and alcohol used technique,initially we large as 1.5–2 cm (refs. ultramicroscopy neurons of subset a in only microscopy. To mice expressing GFP end, this transgenic we used (3DISCO) by optical clearing and light-sheet laser-scanning ultra technique a developed we cord, spinal and brain unsectioned of clearing. tissue To of application achieve imaging high-resolution venuesnew opened for the tissues fluorescentlythe labeled of ing scanning microscopy techniques that can achieve deep tissue imag laser and proteins fluorescent of development decades, recent In 3DISCO Development of resembles glass. thus and transparent, turns and hardens tissue cleared the dure, proce clearing the tissue. impregnated of At the end as the index (DBE) ether clear) Murray’s as known also (BABB, benzoate glucose as such agent, clearing optical an with impregnated is tissue dehydrated Subsequently, compared cellular with containing structures proteins and lipids water of index refractive low the to owing dehydration, tissue is 5 , Caroline D Hojer 2 , 3 13,1 4 , which have approximately the same refractive refractive same the approximately have which , 11,1 6 , which can image organs with dimensions as as dimensions with organs image can ,which 1 1 natureprotocols . However, THF- and BABB-based clearing clearing .However, BABB-based and THF- 11,17–19). the During development the of 1 5 6 and used light-sheet laser-scanning laser-scanning light-sheet used and SCO , Jackson G Egen 7 , glycerol , is a powerful technique that 4 Department of Neurobiology,of Department

| 8 VOL.7 NO.11VOL.7 6 , benzyl alcohol–benzyl alcohol–benzyl benzyl , Department of Discovery of Department 1 0 . alcohol addition,In protocol 6 4,9–1 ,

3 or dibenzyl dibenzyl or | 2012 SCO |

1983 , 4 - - - . © 2012 Nature America, Inc. All rights reserved. cal cal research. can be a universal procedurehistology in many biomedi fields of it therefore, tissues; tumor and glands mammary nodes, lymph worked on every tissue that we have tested, including lung, spleen, cord spinal same the in microglia tion along with the spatial of activated distribution astrocytes and cord microglia and neurons in the same cleared and unsectioned spinal within a tissue. We have successfully labeled and imaged astrocytes, is also possible to differentially label multiple target cells/molecules composition presented aboveand further optimizeitifneeded bychanging incubation times byabout50%(50%increase ifthe clearing isinsufficient or50% decrease ifthe clearing istoo much). results. Forexample, for spinal cord and brain clearing, refresh DBEevery10minand 12h,respectively. To optimizethe clearing for different tissues, follow the protocol for the tissuehaving similarsizeand a mCherry and so and on), dyes synthetic mCherry (CFP,GFP, fluorophores YFP, RFP, of expression transgenic ing 3DISCO on several organs at the same time to perform largeinterspersed screens. between incubation times. Therefore, one can describedbelow), which require onlyfewminutesa actual of work and sectioning during subsequent image problems.alignment distortions mechanical possible of because tissue the entire intact of imaging 3D with compared incomplete be likely most would images reconstructed Moreover, weeks.these several take would and imaging sectioning, lar data using serial ‘stitching’ the data of a day for large organs such as the brain ( meters) such as spinalmilli cords, few (a mammaryorgans small glands for or hours lymph few nodes, a only and takes It sections. cord or blood vessels covering entire organs. environment, e.g., neurons extending throughout the entire spinal their to relation in organs entire within cells/tissues large of logy obtainto impossible was otherwise which cord, spinal unsectioned in glia reactive of distribution spatial the and axons regenerating of trajectories the follow to method We this distribution. used have in cells/molecules large tissues, including their and spatial density and labeled about information accurately histological complete acquire to quickly ability the provides technique 3DISCO The Applications the and method advantages of we tested, including adult brains that tissue every THF resultedthat of in decentwith tion clearing new clearing protocol using DBE—instead of BABB—in combina T 1984 For small tissues, the 70%(vol/vol)THFstepcanbeskippedtosavetime. Changing the clearing solutions abouttwoorthree times during the totalincubation times of 100%THFand DBEhelps toget better able able DBE DCM 100% (vol/vol) THF 80% (vol/vol) THF 70% 70% (vol/vol) THF 50% (vol/vol) THF R protocol 3DISCO is compatible with diverse labeling methods includ methods labeling diverse with compatible is 3DISCO (as steps incubation solution sequential of consists 3DISCO 3DISCO is very fast to perform compared with imaging of tissue eagents 1

1 | , allowing us to study neuronal degeneration and regenera and degeneration neuronal study to us ,allowing VOL.7 NO.11VOL.7 1 1 1 1 . Hence, this method is particularly useful to study the histo |

Clearing protocols for different tissues. | 2012 |

natureprotocols Mammary gland, lymph node 3 3 × 20 min ≥ 1 20 20 min 15 15 min 20 min 20 20 min 4 15 15 min . 1 1 1 1 . Furthermore, 3DISCO has has 3DISCO Furthermore, . and antibody labeling antibody and a Table 1 ). To acquire simi lung, spleen S 3 3 × 30 min pinal cord, ≥ 30 30 min 20 20 min 30 min 30 30 min 15 15 min

easily use 11,2 a 0 . It

­ ­ - - - - - two-photon microscopy. and confocal and epifluorescence wide-field as such techniques a CX3CR1-GFP line labeling microglia labeling line CX3CR1-GFP a half-life of GFP signal in cleared brains (from the GFP-M line the fluorescent signal over time; for example, we observed degrades that the solution clearing final the because periods, prolonged for stored be cannot tissues cleared addition, In tracing. for used microscopy, nor is it amenable to any lipophilic dyes such as those electron with studied further myelin,be cannot tissue cleared the solves the in lipid the structures tissue such as cell and membrane dis clearing optical Because tissues. fixed on used be only can it tissues, the of composition chemical the alters clearing because There are technical and practical limitations of 3DISCO. Essentially, Limitations addition, bear in mind that labeling the entire depth of the tissue tissue the of depth entire the labeling that mind in bear addition, In transparency. their disrupts tissues cleared the of rehydration because immunostaining, the of completion the after applied be should clearing the used, is labeling antibody if that note to tant impor is However, it imaged. are mice adult from glands, mary be a good alternative when embryos cells or structures with synthetic dyes expression or transfection of fluorescent proteins neurons of set sub a in GFP expressing mice Thy1-GFP e.g., cells, or molecules expresses high levels of a fluorescent protein that labels the desired that model mouse transgenic a with is ratio signal-to-noise high achieve to way best by The clearing. detectable before microscopy readily fluorescent is that fluorophore strong a with labeled tissue. of Choice design Experimental protocols.special especially in adult tissues, challenge, which big require a long incubationis times tissues and large of immunolabeling penetration, antibody limited of Because fluorophore. strong a with done be should labeling 3DISCO, with results best To achieve solutions. clearing in others than stable less are YFP) (particularly proteins soon as possible after optical clearing. In addition, some fluorescent approximately 1–2 d. For this reason, the tissue should be imaged as Finally, microscopy cleared can tissues be using various imaged B rain stem ≥ 2 2 × 1 h 45 45 min 30 30 min 1 1 h 1 1 h 1 1 h a 1 5 ( Fig. Ideally, the tissue being analyzed should be be should analyzed being tissue the Ideally, 1 1 h, overnight, 1h 1 ), a GFAP-GFP astrocytes labeling line B ≥ 1 1 h 1 1 h 1 h rain — 3 3 h 2 2 2 or soft tissues, such as mam 5 . Antibody labeling can also 2 2 . Alternatively, use viral viral use Alternatively,. B rain (longprotocol) 23,2 3 3 × 12 h 1–2 1–2 d 4 12 12 h 12 h 12 12 h , or labeling of — 1 2 5 1 ) ) is or - - - - © 2012 Nature America, Inc. All rights reserved. (reducing incubation times of diluted THF steps) might increase increase might steps) THF diluted of times incubation (reducing steps dehydration gradual reducing that mind in However, bear time. save to directly) THF (vol/vol) 80% to THF (vol/vol) 50% from (change skipped be can step THF (vol/vol) 70% and the easy, fast relatively cleared be can tissues small such As h). (1–2 times incubation shorter requires cord) (spinal tissue smaller a DBE, and THF 100% for especially h), (12–24 times incubation has a sturdy composition with myelin (e.g., brain) requires longer ness and the sturdy nature of the tissue. Whereas a large tissue that In general, are times the optimum incubation on based the thick cleared. being are tissues large when overnight or hours few a to or for a few hours. The final step clearing in DBE can be extended overnight done be can steps THF 100% The flexible. is somewhat step each of duration the general, In pipette. Pasteur a using vials glass in solutions of changes sequential requires only which clearing. Tissue preferred labeling. waytissue of the not is immunostaining time, the of most Therefore, tissues. (PFA)-fixed paraformaldehyde in labeling good yield antibodies all not addition, In tissues. the within antibodies the of etration techniques and long incubation times, because of the limited pen with zoom: ×1.6 to ×0.8 magnification on ×2.0 lens, scan duration: ~1 h 30 min). ( mice using ultramicroscopy. ( Figure 1 institutional guidelines for the use of laboratory animals. The Genentech Institutional Animal Care and Use Committee approved all planemouse in protocols. 2 depths marked in µ a Box 1 in and indicate whether the chemical istoxic ornot. Find the hazards and handling information of allthe chemicals usedinthis protocol dilutions of THFsolutions canbecollected into one bottle. Labelthe wasteclearlywiththe date, content, producer and lablocation, Collect allof the clearing solutions into separate wastebottles(made of glass)and store the wasteinafume hood. Different

m, the number of optical sections: 1,051, zoom: ×5.0, scan duration: ~1 h). ( 1 mm T

immunostaining may require special immunohistochemistry able

e | . ( Typical results obtained from 3DISCO of neurons in the brain and hippocampus. We imaged entire cleared brains and hippocampi from GFP-M line

2 g ) Higher magnification of the region indicated in . Followthe relevant institutional rulesfor using and discarding waste material and chemicals. | Waste handling a . ( Tissue clearing is a straightforward procedure, straightforward a is clearing Tissue e ) 3D reconstruction of a cleared hippocampus with a corner-cut view showing some of the neurons that reside in the tissue ( a

) 3D reconstruction of an entire mouse brain with a corner-cut view (

d c

f

. The fixed mouse brains and hippocampi were collected in accordance with governmental and b b e - -

b f – f d ) The horizontal projection from the cleared hippocampus at the indicated Box important that they be handled and disposed of appropriately (see small tissues. for particularly the of clearing pared with BABB com agent clearing optical superior a is DBE times. incubation fluorescent signal. Hence, we use it only for small tissues with short Dichloromethane is a good lipid-solving agent, but it also decreases clearing is insufficient or 50% decrease if the clearing is too much). the if increase (50% 50% about by times incubation changing by in a tissue having similar size and composition to the one we present terresults. Hence, protocol followthe to first users for we suggest the total incubation times of 100% THF and DBE helps obtain bet during times three or two about solutions clearing the Changing the tissue shrinkage and distortion due to the fast removal of water. There are other light-sheet scanning techniques that can be used: used: be can that techniques scanning light-sheet other are There microscope. confocal a of price the half about costs and available scope adult rodent brain, we use a light-sheet laser-scanning ultramicro the as such organs To procedure.large image imaging the is step 3D imaging of cleared tissues. ) The horizontal projections (~100 Many of the reagents used for clearing are toxic, and it is therefore Table 1 1 and 0 . The ultramicroscope has recently been made commercially 1 . The protocol can then be further optimized if needed needed if optimized further be then can protocol .The Table 1 mm z -step: 3 2 1 4 for information). further c , but BABB can be used as an alternative to DBE natureprotocols µ m, the number of optical sections: 2,518, f µ After the tissues are cleared, the next m) from the cleared brain at different g

| VOL.7 NO.11VOL.7 d 200 protocol µ m | 2012

g z -step: |

1985

- - - © 2012 Nature America, Inc. All rights reserved. we use the 12- or 16-bit data for quantification. sufficient, not resolution is 8-bit to analyzed. be scans good If the select and Amira of view multiplanar the in samples the examine Amira, convert the data from 12- or 16-bit images into 8-bit images, algorithm. tion sections), on which it could take 1–2 d to run a robust deconvolu optical (4,000 data of GB ~40 produces it brain, mouse adult of 3DISCO acquire we when example, For deconvolution. as such it may take hours to several days algorithms to advanced perform can size that file be obtained the enormous sible, but considering sues are imaged. the Improving data sets image with filters is pos tis large very if systems computing powerful require may it and Neurolucida or used. be can Imaris Alternatively, quantification. segmenta and tion visualization, for Amira use We software. imaging 3D available commercially with or ImageJ as such software free analysis. Data quantification. and for analysis data subsequent information illumination possible maximum the obtain to prefer acquiring images with a pixel intensity range of 12 or 16 bits versus 10–20 mm). For of microscopy all using 3DISCO,types we 0.2–2.2 depth: imaging mm; 10–20 versus 0.5–1 area: (imaging area and depth will be small compared with light-sheet microscopy imaging maximum the techniques, microscopy two-photon and high–numerical aperture (NA) objectives that are used for confocal of the relatively and high magnification small working distance of only be reached when a cleared tissue is imaged. However, because tice, by reported resolutions can optimal manufacturers objective microscopy to obtain extremely high resolution scanning confocal microscope photomultiplier with tubes. point- a is it because ultramicroscope an with compared slower is and resolution lower times four or three delivers it but brain, rodent the as large as tissues image can which microscope, LCI) alternative is the true confocal scanning–large scale imaging (TCS- microscopy illumination plane selective microscopy fluorescence light-sheet laser scanned digital protection isrecommended tominimizethe inhalation of clearing reagents. Allstorage containers including the wastecontainers should beglassand stored inthe dark. For handling organic solvents, usealabcoat,nitrilegloves(8mil,double glove),splashgoggles and perform allthe experiments under achemical hood except the imaging step.Usage of proper respiratory T 1986 able able Paraformaldehyde Benzyl benzoate Benzyl alcohol Dibenzyl ether Dichloromethane Tetrahydrofuran C protocol hemical name Our typical workflow in data analysis is to load the data into into data the load to is analysis data in workflow typical Our Analyzing the data is the most time-consuming step of 3DISCO, confocal or two-photon by imaged be also can tissues Cleared

| VOL.7 NO.11VOL.7 2 2 |

Reagent safety information. Analysis of 3DISCO data can be achieved with with achieved be can data 3DISCO of Analysis | 2012 | natureprotocols Flammable Yes Yes Yes Yes No No 1 7 . Another commercial commercial Another . 1 C 1 . Indeed, in prac orrosive No No No No No No 2 6 and and - - - - -

T oxic Yes Yes Yes Yes Yes Yes 3,000–4,000 optical images (16-bit images with a 5.5-megapixel 5.5-megapixel a with images (16-bit images optical 3,000–4,000 a 30- to 40-GB data set at 2–3 approximatelyproduces ultramicroscopy by line) GFP-M mouse sets. (from data the scan For large brain example, GFP adult each produce used, microscopywill is two-photon or ultramicroscopy Image collection, storing and backing up. (Nvidia Quadro (Nvidia 5000) and 196 GB RAM. of card graphics powerful a with equipped workstation, Quad-core HP a GB, used 30–40 we as large as sets data RAM.Foranalyzing HP workstation with a dual processor and equipped with 16 GB of an used we GB, 1–3 around sets data analyzing For imaging. the power required is correlated with the size of the data collected from ing than rather postprocessing.time and imaging The computing is best to optimize the experimental steps, including labeling, clear Computing power, data analysis. done carefully and systematically. be should backup data and storage collection, image Therefore, Edge camera). The data can grow to a few terabytes in a short time. (iii) (iv) Important points Important to keep in mind are as follows: (ii) (v) (i)

Usefastest possible the connectionsexternal storages with Never collect the data to a network drive imageduring ac If any of the storage media carrying the data fail,data the immedi carrying storage the media any of If ately create copy. another full connection. to transfer and analyze the data, such as through an eSATA above.mentioned as created are data the of copies two least at from lost. being networkthe drive safestwaythe is data to protect data the drives), network than quicker much drive external from data the (computers load quickly data the analyze to used safe network drive. Whereas the external drive copy can be a to one and storage external an to one copies,two make disrupted. be will scan the shortage, power or service maintenance as such reason, any for fails connection network the If quisition. Delete the data from the imaging computer only when when only computer imaging the from data the Delete When the data are successfully collected to a local drive, local a to collected successfully are data the When O xidizer No No No No No No Form peroxides µ m m To reduce the processing time, it z -resolution, which yields about Yes Yes Yes No No No 3DISCO, especially when R eactive No No No No No No - - - © 2012 Nature America, Inc. All rights reserved. institutional guidelines for the use of laboratory animals. The Genentech shown in this figure were collected in accordance with governmental and of clearing, the tissues should look transparent. The fixed mouse brains with a Pasteur pipette and add the next clearing solution. ( until the end of incubation time. ( rotating wheel ( ( organs into glass vials and immediately fill with the first clearing solution. vials (2–3 ml), which are easy to open and close. Transfer the postfixed in visibly labeled glass bottles. ( Figure 2 • microscopesImaging • • • • • • • • • • • • • EQUIPMENT • • • • Tracing stainingreagents andantibody • • • • • reagents Clearing • • • • • • • • • Fixative reagents andperfusion • Tissue REAGENTS M Institutional Animal Care and Use Committee approved all mouse protocols. c Benzyl benzoateBenzyl (Sigma-Aldrich, cat. no. B6630-250ML) alcoholBenzyl (Sigma-Aldrich, cat. no. 305197-100ML) DBE (Sigma-Aldrich, cat. no. 108014-1KG) Dichloromethane (DCM;Sigma-Aldrich, cat. no. 270997-12X100ML) THF (Sigma-Aldrich, cat. no. 186562-12X100ML) PBS (Sigma-Aldrich, cat. no. P3813-10PAK) NaOH (Mallinckrodt, cat. no. 7708-10) HCl (Fisher Scientific, cat. no. A144-500) (Braun,Saline cat. no. S4011-S) 2-Methyl-2-butanol ( 2-2-2 Tribromoethanol (Avertin, Sigma-Aldrich, cat. no. T48402) PFA (Sigma-Aldrich, cat. no. P6148) NaH Na and Accreditation Laboratory of Animal Care (AAALAC)-accredited facility. Use Committee and were carried out in an Association for the Assessment guidelines and principles set by the Genentech Institutional Animal Care and animal experiments in this article were approved by and conformed to the tutional regulations regarding the use animals of for research purposes. All must be handled in accordance with the relevant governmental and insti we anultramicroscope used from Lavision Biotech or theLeica TCS-LSI In thisstudy, large tissues(size bigger than0.5mm), of for theimaging Fume hood Quantofixperoxide (Sigma-Aldrich, test strips cat. no. 37206) Dental cement (LangDental, cat. no. 1223PNK) Glass Petri dish(Corning Pyrex, cat. no. 3160-101) Coverslip (Corning, cat. no. 2940-245) FastWell (Grace Bio-Labs, cat. no. FW20) Microscopy slide (VWR, cat. no. 48311-703) medium flow (Gilson, pumphead cat. no. F117606) Minipuls evolution pump(Gilson, perfusion cat. no. MF4 F110701)with Kimax storage media (100ml; bottles Cole Parmer, cat. no. EW-34523-00) Aluminum foil (Fisherbrand, cat. no. 01-213-104) Mini rotator (VWR, cat. no. 100498-878) Forceps (FST, cat. no. 11251-10) (VWR, cat.Glass vials no. 548-0554) Alexa IgG Fluor (Molecular anti-rat 488goat Probes, cat. no. A-11006) cat. no. C6198) Mouse conjugated muscle antibody anti–smooth actin to cy3(Sigma, CD24(BDPharmingen, anti-mouse Rat cat. no. 553146) FITCLectin (Sigma-Aldrich, cat. no. L0401-1MG) at NYU); and C57BL/6 (Charles River) sity); CD11c YFP-Venus (Rockefeller University); CX3CR1-GFPKras (Littman lab (Washington University in St. Louis); Thy-1following GFP (line mice founder are examples 1, Genentech); of suitable animals totissues use: Thy-1 can GFP (GFP-M)be labeled line with viralAnimal expression, with strong synthetic fluorescence dyes or expression antibody labeling. in The the desired tissue Alternatively, the , ATER d ) ) Place the glass vials with the tissue and clearing solution on to a 2 HPO LSLG12D 2 PO I

| ALS 4 4 Clearing reagents and steps. ( (Mallinckrodt, cat. no. 7892-04) (Mallinckrodt, cat. no. 7917-16) mice (Jacks lab at MIT); MHCII-GFP (MIT and Harvard Univer c ) ) and keep the samples in the dark ( tert -Amyl alcohol, Sigma-Aldrich, cat. no. 152463) b ) ) Perform tissue clearing in small glass e ) ) Next, remove the clearing solution !

a CAUT ) ) Prepare the clearing reagents I ON The experimental animals d ). ). Rotate the samples f ) ) At the end

- - (21.8 g) NaHand (21.8 REAGENT SETUP • • Image analyzingtools • • • • Leica LSIsystem with equipped • • • • • • • • Biotech) with Ultramicroscope (Lavision equipped (total) 0.1 M PB. of Heating the 0.1 M PM solution to 50–60 °C and addition PFA (4%, vol/vol) mouse. of weight body of gram per mg ~0.5 is dosage The use. before temperature room at dissolved re- be to has Avertin The solution. the harm not will this but °C, 4 months. 2–3 to up for °C 4 at stored be can solution working (vol/vol) 2.5% year,and 1 to up for °C 4 at stored be can solution stock 100% The PBS. ×1 or saline sterile in 1:40 Avertin 100% dilute solution, working (vol/vol) 2.5% a pare (total) of ml 10 Avertin at room temperature (18–25 °C) for up to 6 months. For 0.1 M PB, dilute 0.2 M PB 1:1 in distilled water. This solution can be stored buffer Phosphate (PB; 0.1 M) and 0.2 ImageJ (freeware from theUSNational Health Institutes (NIH)) of ResolveRT with Amira microscopy module(Visage Imaging) Excitation lasers (405, 477, 532and635nm) Filters for DAPI, FITC andrhodamine PLANAPO 5.0×/0.5LWD PLANAPO 2.0×/ WD 39mm FITC filters andrhodamine PCO Edge SCMOSCCD Coherent sapphire laser 568 Coherent sapphire laser 488 Biotech) reservoirImaging resistant to (100%quartz) chemicals clearing (LaVision Biotech) Lens resistant plastic) cap(black to delrin chemicals clearing (LaVision MVPLAPO ×2lens Olympus binocularbody (MVX10) Olympus manufacturers used. canbe SP5 confocal system, but any confocal or multiphoton system from other upto 0.5mm), of multiphoton weview anOlympus used system or Leica aswell. used can be resolution For animaging tissuesathigh (with imaging confocal microscope, but illumination other microscopes light-sheet with e c a

To prepare 100% Avertin solution, dissolve 10 g of Avertin in in Avertin of g 10 dissolve solution, Avertin 100% Toprepare  tert 2

PO

CR To prepare 4% (wt/vol) PFA, dissolve PFA 40 g of in 1 liter -amyl alcohol by shaking it at 37 °C overnight. To pre overnight. °C 37 at it shaking by alcohol -amyl 4 I (6.4 g) in 1 liter of distilled water. distilled g) in 1 (6.4 liter of Adjust pH the to 7.4. T I CAL natureprotocols Avertin precipitates out when it is stored at at stored is it when out precipitates Avertin

Prepare 0.2 M PB by Namixing f d

b | VOL.7 NO.11VOL.7 protocol | 2012

| 2

HPO 1987

- 4

© 2012 Nature America, Inc. All rights reserved. 5| pipette ( the firstclearing solution (order isaslistedin 4| T ­placing the tissuesinaPetri dishfilledwithPBSand removing the extra tissueswith forceps under abinocular stereoscope. 3| organs are taken from aged animals).  shortened toafewhours, especially for small tissuessuch asspinal cords ormammary glands. 2| ? ­regulations regarding the useof animals for research purposes. ! completely removed from the tissue. Alternatively, ×1PBSsolution canbeusedinstead of 0.1MPB. The perfusion time canbeshortened aslong asthe bloodis using 0.1MPB(PB,pH7.4)for 5–10minfollowed by30–40minof 4%(wt/vol)PFA in0.1MPBatarate of 3mlmin 1| T PROCE avoid toxic; is solution working THF lid. compatible a with bottle glass ! weeks. 2 than more for solutions working same the use not Do ture. ( clearly them label and bottle glass different a in solution working THF each Prepare minutes. few a for it shaking by mix and bottle glass 100-ml a in water distilled of ml 50 and THF of ml 50 add solution, working THF 50% for example, water. For distilled in solutions working THF (vol/vol) 80% (vol/vol)) 80% and 70 (50, solutions working THF the desired volume from thebottle. volumes are (e.g., used 0.5–1 liter), through thelidto remove useasyringe a fresh theworking time stock solutions are every prepared. larger stock If stock solution assmall-volume units(e.g., to 100-mlbottles) able to be open oxygen.of covered canbe the bottles such gas asargon to aninert prevent with access (e.g.,tion, thelarge THFbottles 0.5–1liter) are if stocked for longer times, com/chemistry/so lines for peroxide-forming compounds carefully ( peroxidelevels with before test strips usingtheoldstocks. Follow theguide to years, itmay peroxides, form explosive. which canbe Check theperoxide THF( handling when avoid skinandeyes. inhalationor contact with Use proper gloves andamask acompatible lid. with bottle andinaglass THF inafumehood THFistoxic; use thesameworking solutions for more than2weeks. Store thesolution inthedark, sealed. tightly To results, thebest obtain do not (100 ml)to avoid repetitive stock bottles. opening of clearly. thebottles Label THF (99–100%) waste material andchemicals. ! Use proper gloves andamaskto PFA handle andNaOH ( toxicis avery reagent. Avoid skinandeyes. inhalationor contact with use as a fixative canbe stored at 4 °C for up to 1–2 months. pH to 7.4 using HCl. Finally, filter the solution in a chemical hood. PFA for a few NaOHof pellets help dissolve PFA faster. NaOH If is added, readjust the 1988  Fig. 2 Fig.

protocol

issue preparation issue issue clearing CAUT CAUT

CAUT TROUBLES

CR CR

Close the lid of the glassvial and placeitonto aturning wheel ( Transfer the organs from the postfixation solution (4%(wt/vol)PFA) directly into the glassvial, and immediately add Remove the extra tissues(connective tissue, meninges ordura mater) surrounding the organ. Thiscanbedone by Dissect the organs of interest and postfixthem by immersion in4%(wt/vol)PFA overnight. Thisstepcanbeskippedor Anesthetize the animal using 2.5%(vol/vol)Avertin (0.5mlper25gbody weight intraperitoneally) and perfuseit | a I I I VOL.7 NO.11VOL.7 I ). Store the solution in the dark, tightly sealed, at room tempera room at sealed, tightly dark, the in solution the Store ). ON T T ON D I I I ON URE CAL CAL  Follow for therelevant institutionalusinganddiscarding rules Fig. 2 Prepare THF working solutions in a fume hood and in a in and hood fume a in solutions working THF Prepare

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- - © 2012 Nature America, Inc. All rights reserved. cannot collecthigh-resolution images, particularly inthick tissues. The stepsdescribed below(option C)canalsobeusedin tissue. Becausethe fluorescence excitation light willstimulate the entire samplefrom toptobottom,these imaging methods imaged using fluorescent wide-field orbinocular microscopy (option C). This allowsaquick exploration of the cleared samples canbeimaged with confocal ortwo-photon microscopy asinany other sampleimaging. Cleared tissuecanalsobe or two-photon microscopy, the tissueshould bemounted onaglass slide withacoverslipasdescribed inoption B. Mounted although the scanfieldwill be relatively small compared withultramicroscopy. To be abletoimage the tissueusing confocal cleared tissuescanalsobeimaged using confocal ortwo-photon microscopy toobtainhigh-resolution scans (option B), microscope belowcanreadily bemodified ifanother light-sheet illumination microscope withalarge scanarea isused. The 10| Imaging the cleared tissues immediately after proper clearing is achieved. The sample can still be imaged the next day, but the signal will be weaker.  uncleared tissuein in size uncleared versuscleared brain isshown in 9| ? incubation for adult mouse brain) ( spinal cord tissue, which isrelatively thin(1–2mm), larger organs may require longer DBEincubation (e.g., 6htoovernight should beoptimizedaccording tothe thickness and typeof the tissue. Whereas 15–20minDBEincubation issufficient for 8| not tospillsolutions ifthere are many samplesonthe wheel atdifferent angles. Pasteur pipetteand restart the wheel ( priate wastecontainers (kept inahood). Immediately add the next clearing solution (seeorder in 7| tive incubation times. This stepisthe same for allthe clearing steps. The onlydifferences are the clearing solution inthe glassvial and the respec for the indicated time points ( wheel atconstant speed(20–40r.p.m.) the dark. Rotatethe samplesusing the foil ( 6| approved all mouse protocols. Institutional Animal Care and Use Committee for the use of laboratory animals. The Genentech with governmental and institutional guidelines shown in this figure were collected in accordance the imaging software. The fixed mouse brains view to the camera and adjust the fine focus using the binocular view. ( the screw (red arrows). ( arrows). ( onto the sample holder using the screw ( (white arrow). ( is filled with the final clearing solution, DBE reservoir of sample holder on the ultramicroscope 1 mm. ( the tissue are preserved after clearing. Scale bar, that the overall structures and proportions of view imaged by ultramicroscopy ( with optical projection of a cleared brain in coronal imaged by wide-field fluorescence ( ( brain before ( Figure 3 laser-scanning wide-field microscopes such asthe Leica LSI,which cancollecthigh-resolution image stacks. c

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Image tissues Proceed toimaging assoon asclearing issufficient. The cleared tissueswillbetransparent and hard (an example of Repeat Step7until the lastclearing solution (DBE)isadded. To achieve agood transparency, DBEincubation time When the time for the current clearing stephaspassed, stopthe wheel and discard the clearing solution into appro Cover the sampleswithaluminum Fig. 2 e 1

) The ultramicroscopy setup. ( | 1 i Ultramicroscopy setup. ( , the proportion and general anatomy of the structures are preserved incleared tissue(see ) The laser will travel perpendicularly to

PO a d ) and after clearing ( g ) tokeep the samplesin H I , NT h OOT ) Mount the cleared tissues k The DBE solution degrades the fluorescent signal over time. Therefore, the best result is obtained by imaging ) Subsequently, switch the . Forimaging large tissues, weuseanultramicroscope ( I j N ) Adjust the focus using Fig. 3 G d d ) demonstrating ). c a b ) compared , ). b ) A mouse ● f

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| VOL.7 NO.11VOL.7 c 1 compared with ) using afresh protocol | 2012

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- 1989 - © 2012 Nature America, Inc. All rights reserved. ( ( ? 1990 (xvii) Startthe actual scan. (viii) Stopthe scanand switchthe view tothe microscope bypulling outthe controller. (xiii) (xvi) (xiv) Move the protocol B A (xii) (vii)

(xv) Stopthe scanand define the (iii) (iii) Placethe sampleholder withthe tissueinto the imaging reservoir withthe DBEsolution ( TROUBLES (vi) (iv) (xi) Stopthe scan. (ix) (vi) (iv) (ii) Placethe tissueinthe middle of the poolbydirecting the surface tobeimaged upward ( (ii) ) Imagingthetissueswithconfocal ortwo-photon microscopy ) (v) (x) (v) (i) (i) U

| VOL.7 NO.11VOL.7 ltramicroscopy imaging Move the tissue using the ( holder (  of the stage tofind the startposition of the scan. Mark the position asstart.  Alternatively, FastWell glassslides canbe used.  the sliders. If the leftand right sides of the sampleare not evenlyilluminated onthe merge image, adjustthe focus againusing the view scan.Inaddition totwoseparate images coming from right and leftscans, amerge image should appear. until youseeacomplementary image. Stopthe scanafterthe adjustments. Activate the scanonbothsides and start until the image lookscleanand focused. Next, stopthe scanand choose the other side and do the same adjustments First, choose onlyone side, leftorright, and then startthe scaninview mode. Drag the focus slider of the chosen side addition, change the  the scan.Note thatthe maximum that when the slide isplaced onthe sampleholder of the microscope, itdirectly grasps the slide and not the coverslip. ! objective tipand focus point onthe tissue). maximum possibleimaging depth isequal tothe working distance of the usedobjective(the distance from the gap betweenthe coverslipand tissueshould beminimal inorder tobeableimage the tissuedeeper, becausethe power, 100%;attenuation, 100%;camera, 200–400Hz;autosaving directly tothe hard drive. ! After focus and laseradjustments, define the scanfield. In the view mode, change the If anultramicroscope systemwithboth-side laserillumination isused, align the leftand right laserasfollows. To adjustthe fine focusing, startthe scaninview mode and find the best focususing the fine focus handle on the Adjust the optimum scansettings. We usedthe following parameters for imaging cleared adult mouse brains: laser Lower the lens until youseeaclearfocus through the eyepiece of the microscope ( Dip the lens into the imaging reservoir slowly. From the imaging software, choose the ‘no filter’option and startthe laserscaninview mode. Gently mount the sampleusing the screw onthe sampleholder ( Fill the imaging reservoir withthe final clearing solution, DBE( Set the Place the slide withthe sample under the two-photon orconfocal microscope. Use the mounting medium required for Gently applypressure on the coversliptopushitclose tothe tissueuntil the coversliptouches the tissue. Ideally, the When the dental cement hashardened enough butnot completely, fillthe poolwithDBEand coveritwithacoverslip Use dental cement orglasspaint (natural color)to create apoolatleastasthick asthe tissue( Name the scan.Forconvenience during image analysis, include the following inthe title:zoomfactor, 2 line usedand date of the scanifthe software does not automatically keep thisinformation. For example, sample1_2×_ using anoil-immersion objective, asthe refractive index isclosertothatof DBE(refractive index ~1.56). the objectivebeing used( different magnifications.  microscope ( Fig. 4a

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Pay attention tothe direction of the laserscanand align the tissueaccordingly onthe sample natureprotocols Dental cement ispreferable toglasspaint, becauseDBEdissolvesmost glasspaints. Leftand right sides should havecomplementary scanviews. Use lowlaserpoweratthis point inorder toavoid bleaching the samplebefore the real scan.In Before adjusting the focus, choose the desired magnification. The fine focuswillbedifferent for k ). z

-position from time totime (aboutevery30s)iffocusing takes time. x- , Fig. y- and z -steps of the scan. 4 c ): oil(refractive index ~1.515)orwater(refractive index ~1.33).We recommend z z -stage controller of the microscope until the laser sheet goes through the sample ( -thickness of the scanislimitedbythe working distance of the objective. Hence,

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i ). © 2012 Nature America, Inc. All rights reserved. Troubleshooting advice canbefound in ? ( Committee approved all mouse protocols. Genentech Institutional Animal Care and Use guidelines for the use of laboratory animals. The accordance with governmental and institutional tissues shown in this figure were collected in glass Petri dish filled with DBE. The fixed mouse with a wide-field fluorescence microscope using a ( microscope using proper immersion for the lens. with an upright confocal or two-photon glass coverslip. ( the final clearing solution (DBE) and cover with a (white arrowheads) and then fill the pool with the cleared tissues in the middle of the pool border using glass paint or dental cement, place mounted on a glass slide. ( microscopy. ( can be imaged using confocal and two-photon high-resolution images, the cleared tissues confocal and two-photon microscopy. To obtain Figure 4 (viii) d C (vii)

(iii) Placethe glassPetri dishwiththe tissueunder the microscope ( ) Alternatively, cleared tissues can be observed TROUBLES (iv) (ix) Startthe scan. ) Imagingthetissuewithwide-fieldfluorescence orbinocularmicroscopes (ii) (v) Make snapshots atdifferent focal points. The focus range should beimproved when cleared tissuesare observed. (x) (i) FillaglassPetri dishwithDBEsolution ( solution. To avoid spilling the DBEsolution when the dishismoved around, do not fullyfillthe Petri dishwithDBE.  can beproduced bythe objectivethatisused. signal reduction indeeper regions. Check whether there issuch afunction of the software thatyouuse. different acquisition parameters (laserpowerand gain) atdifferent imaging depth, which helps compensate for the might beincreased asthe scangoes deeper and deeper. The Olympussoftware (Fluoview) thatweusedallowsdefining guidelines listedintroubleshooting. Ifathick tissue(e.g., 2mm)isimaged byatwo-photon microscope, the laser pared withuncleared tissues;hence, samplebleaching isnot acommon issue. Incaseof bleaching, follow the  a glassPetri dishfilledwithDBEas described above. working distance. However, asthey do not touch the samples, samplemounting isnot required. They canbeimaged in focal microscopy withaworking distance of 4mmand 100 two-photon microscopy withaworking distance of 2.2 mm,and ×10(0.25–NA, Leica) or×40(1.25–NA,Leica) for con that isused. Typically, weused×20(0.95–NA,Olympus)or×25(1.05–NA, Olympus)water-immersion objectivesfor working distance of the lens mounting onlyasthick asthe samples withdental cement for convenience, prepare tissue Turn onthe shutter of the arc lampand observethe tissuewithalow-magnification airobjective. Adjust the laserpowertoaslowpossible. Imaging of cleared tissuesrequires considerably lesslaserpowercom Place the cleared tissueinthe middle of the Petri dish( If needed, scanoverlapping regions (atleast10%overlap)of the samplewithconfocal ortwo-photon microscopy to Define the samples for imaging assoon aspossible. and mount the pieces asdescribed. As DBEdegrades the fluorescence signal overtime, itisbesttoprepare the mount, orifthe region of interest isdeeper thanthe possibleimaging depth, simplychop the sampleusing ablade focal ortwo-photon microscopy bymounting them onaglassslide asdescribed above. Ifthe sample istoobig to  are equippedwiththisfunction. Amotorized stage might beneeded toperform tiling automatically bythe software. be abletoimage alarger area. Thisiscalledtiling or stitching, and many commercially availableconfocal microscopes

| 3DISCO with wide-field fluorescence,

CR CR CR a ) To this end, the tissue should be I I I H T T T c I I I OOT ) Image the slide preferentially CAL CAL CAL z I -step sizeonthe software. Use the optimum resolution of the objective, which isthe bestresolution that

N STEP STEP STEP G b ) First, create a thick DBEsolution should beslightly abovethe cleared tissue;ifthisisnot the case, add more DBE Use onlyairobjectives. The objectiveshould not touch the DBE,which canharm it. Note thatcleared samples, which are imaged byultramicroscopy, canbefurther imaged withcon T able a d 3 Fig. . 4 d ). Fig. µ 4 m, respectively. The airobjectiveshavealonger range of d ). Fig.

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| VOL.7 NO.11VOL.7

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|

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| VOL.7 NO.11VOL.7 3 3 | Data Data are too large to analyze The image is blurry fluorescence signal Weak or no detectable fluorescence signal Weak or no detectable transparent The tissue does not get P

Troubleshooting table. roblem | 2012 | natureprotocols

File File format is not optimal Low computing capacity Unnecessarily high acquisition Large file size due to 12- or 16-bit image The focus is not correct Peroxidized clearing chemicals Insufficient laser power tracers are used) Inefficient labeling (if synthetic dye used for labeling) Inefficient labeling (if antibody staining is expression is used for labeling) Low fluorescence expression (if viral models are used for labeling) Low fluorescence expression (if transgenic Excessive clearing Insufficient clearing Blood is retained in the tissue P ossible reason z -resolution

antibody concentrations and check the Increase the incubation times and/or the labeling without clearing Increase the virus titer and check the efficiency of method to obtain a brighter signal detectable, use another transgenic line or labeling them without clearing. If the signal is not clearing by making tissue sections and imaging Check whether the signal could be detected before especially the final DBE step Reduce the incubation times of the clearing steps, especially the DBE step Extend the incubation time of the clearing steps, initial wash out solution during perfusion wash out the blood from the tissue. Add heparin to Use proper fixation methods (cardiac perfusion) to S optimal format convert the acquired format (e.g., tif) into the acquire the images in this optimal format or image Learn the optimal file format for the gigabytes of RAM (at least 16 GB) Use dual- or quad-core workstations with several this value the lens used and set the Check out the maximum possible convert 12- or 16-bit files into 8-bit files will be sufficient most of the time. Alternatively, Capture images at 8-bit resolution. The 8-bit size sample Zoom 1:1 at the image and readjust the focus on remove excessive peroxide as described using peroxide test strips. Use fresh reagents or ­concentrations. Check peroxide concentrations ­diminish fluorescent signal even at very low Peroxide contaminations in THF or DBE can the exposure time of the image capture Increase the power of the imaging laser. Increase only when sufficient labeling is achieved imaging without clearing. Proceed to clearing efficiency by making some tissue sections and Improve the dye labeling protocol. Check the antibody labeling Look for dedicated efficiency of the labeling without clearing. olution - analyzing software that is used. Either 11,2 protocols for deep-tissue ­ 0 z -resolution at most to z -resolution of

1

4 (continued)

© 2012 Nature America, Inc. All rights reserved. example,integration of transplanted any applied to be tissue. can For S theentire organsin tion resolu high at capillaries of blood visualization allows method also This data). unpublished andM.S., (A.E. TBI after brain and Institutional Animal Care and Use Committee and Use Care Committee Animal Institutional The Genentech animals. of the use laboratory for guidelines and institutional governmental with accordance in collected were cords and spinal brains mouse The fixed min). ~30 duration: lens, scan ×2.0 on magnification ×3.2 to ×6.4 zoom: 616, sections: of optical ( rendering volume map ( code color- in shown vasculature cord the spinal ( h). ~1 duration: lens, scan ×2.0 on magnification ×0.8 to ×1.6 zoom: 1,851, sections: of optical the number ( map ( color-code in shown of vasculature the brain reconstruction ( FITC ultramicroscopy. by and imaged labeled was The vasculature cord. and spinal the in brain of vasculature 5 Figure micrometers ( few of a resolution a at andthehippocampus entire brain mouse scan Notably,to us allowed 3DISCO thetissue in distribution andmap their bodies andcell quantifyglia detect automatically to Wealgorithms developed ( axonsmm 5 over injury that canbeachieved bythe various microscope systems. scatter and canpenetrate through the entire tissue. Thisallowsacquisition of fluorescent signals atthe highest resolution imaged inthe final clearing solution, which hasthe same refractive index asthe cleared tissue, the imaging light does not 3DISCO isbasedonoptical clearing of tissuestomatch the refractive indices throughout the tissue. When cleared tissues are ANT Step 10,imaging the cleared tissues:1–2h Steps 4–9,tissueclearing: 3 hto3d Steps 1–3,tissuepreparation: 1htod ● T approved all mouse protocols. mouse all approved b able able S S upplementary Video 4 ) and volume rendering ( rendering ) and volume

tep 3DISCO is a versatile method versatile that a 3DISCO is We have used 3DISCO to study to neuronal cord3DISCO spinal degeneration Wereactionsused after cell gliahave andregeneration, as well as upplementary Video upplementary T IMI I S C upplementary Videos 2 2 Videos upplementary 1 I 3 3 1

PATE and traumatic brain injury (TBI) (A.E. and M.S., unpublished data). We data). thethelength quantified of regeneratingunpublished andM.S., (A.E. (TBI) andtraumatic injury brain N | | Typical results obtained from 3DISCO 3DISCO from obtained Typical results Sample Sample bleaching P G d

Troubleshooting table (continued). roblem ), transparent grayscale ( grayscale transparent ), D RESULTS d – f a ) ) ( f ), transparent grayscale grayscale transparent ), 1 ) 3D reconstruction of ) reconstruction 3D 1 z . For tracing For . of axons the in spinal cord, the mostly used semiautomatedwe filament-tracing Amira in tool -step: 2 -step: c ) ) ( in vivo in ) to be assessed.

1 2 z 7 ). 3DISCO also allowed us to assess the density and distribution of glia cells after the lesion. thelesion. after of cells glia thedensity anddistribution assess to us allowed also 3DISCO ). -step: 3 -step: µ ( m, the number the m, number Fig. Fig. with lectin lectin with e a and ) ) and – 5 µ c ) ) 3D m, m, ), permitting details of vasculature in the brain ( the brain in of vasculature details permitting ), 3

Repetitive Repetitive imaging Weak sample labeling Laser power is too strong P ). By using these scans, we could analyze the patterns of neurodegeneration thepatterns analyze the could scans, in these we using By ). ossible reason a c 2 mm b tings on the tissue away from the region of interest repetitively. If needed, optimize the acquisition set Avoid imaging the same location on the samples no detectable fluorescence signal’) samples as described above (see Step 10, ‘weak or Troubleshoot to increase the labeling of the the acquisition to compensate Decrease the laser power and increase the gain of S olution Fig. 5a Fig. natureprotocols f 250 d – c µ ) and spinal cord ( cord andspinal ) m

| VOL.7 NO.11VOL.7 e protocol Fig. 5d Fig. | 2012 Fig. Fig.

, 1 e | 1

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© 2012 Nature America, Inc. All rights reserved. with traditional with sectioning tissue methods. wardand reproducible shortdata a various time organs applied histological in to comparedbe can obtain to protocolthat Moreover,applications 3DISCO has clinicalpathology, in cancer suchstaging.Thus, in as extremely an 3DISCO is straightfor indicated indicated regions in ( and microscopy coded by color two-photon CD11c-Venus ( reconstruction of lymph nodes from MHCII-GFP ( respectively. Scale bars ( projections from the indicated regions in Scale bars ( sections: 850, zoom: ×2, scan duration: ~30 min). density; cell coded (blue, color density; low cell red, high GFP ( ( from MHCII-GFP ( subsets. cell in monocyte,primarily macrophage and dendritic line and expressing cells; a GFP CX3CR1-GFP the YFP variant, Venus, in dendriticprimarily and a monocytes; CD11c-Venus line expressing in dendriticprimarily cells, macrophages, B cells expressing GFP as a fusion to endogenous MHCII line II complex (MHCII)-GFP histocompatibility from the following transgenic lines: a major immune in the cells and nodesspleen lymph of immune organs. We and cleared imaged Figure 7 mammaryglands, and examplesofof 3DISCO,the versatility andquick screening ofmodulatevasculature, as that molecules and well tumor as tumorsize location, in tissues.As further hours. obtainedwithin be the Assessment of can vasculature tumor tissues of such crucial information easy should allow can easilybeassessedinaveryshort time ( such induced as tissues stem cellgrafts pluripotent orthe distribution and volume of tumors inlarge organs such aslungs Committee approved all mouse protocols. collected in accordance with governmental and institutional guidelines for the use of laboratory animals. The Genentech Institutionalantibody Animal forCare 60 andh at Use4 °C (a total of 4 d staining protocol). ( The tissues were incubated with the primary (1:500) were used to label smooth muscle cells ( muscle actin antibody directly conjugated to Cy3 epithelial cells ( Fluor 488 secondary antibodies used to label rat anti-mouse CD24 primary (1:100) and Alexa (C57BL/6) mouse were immunostained as follows: cells of an entire mammary gland of a wild-type ~3 min). ( of optical sections: 25, zoom: ×2, scan duration: sagittal view ( (gray) with the tumors (red) in horizontal ( LSI ( the tumor were cleared and imaged using Leica without tumor ( weeks after infection. ( expression, and the lungs were collected 16 2 mg ml inducible GFP lentiviral Cre vector expressing doxycycline- cells. The lung tumors were initiated using a was induced specifically in bronchiolar epithelial Kras of lung tumors and mammary glands. ( Figure 6 1994 and guidelines institutional for the use ofanimals. The laboratory Animal Genentech Care Institutional all mouse and approved protocols. Use Committee z protocol -step: 2 -step: LSLG12D b c

). ( | ) ) mice imaged and using ultramicroscopy VOL.7 NO.11VOL.7

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1 mice, the G12D oncogenic allele of | c | doxycycline weekly to initiate GFP e Typical results obtained from 3DISCO , Typical from obtained results 3DISCO µ d a – m, m, the number of ( duration: bars scan ×25, Scale zoom: sections: ~15 377, min). optical ) 3D reconstruction of the entire lung – h z 2 c ) The epithelial and smooth muscle -step: 2 -step: 8 a d h ), 500 . Mice were treated with or without – ) ( a ) or CX3CR1-GFP ( ) or CX3CR1-GFP e c ) and the infected lungs with ) and mouse anti–smooth ) ) 3D reconstruction of spleens a z ), ), CD11c-Venus ( -step: 203 g µ | 2012 – µ m. ( a i d m, m, the number of optical , , respectively. ( bars Scale , – b f ) The control lungs d ), 25 – Figure | f

µ ) Optical natureprotocols m, the number µ m. ( i ) ) mice imaged b ) ) or CX3CR1- 7 g a –

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j – l ), ), 100 Fig. 6a CX3CR1-GFP CD11c-Venus MHCII-GFP Mammary gland Lung c b a 500 e a g µ – , Control (notumor) m. m. The fixed nodesmouse lymph in and accordance governmental were with spleens collected h µ (CD24 Abstaining) h m ) The merge is shown in shows results from cleared and imaged antibody-labeled whole-mount Epithelial cells – d ; clearing and imaging intotal3–4h).Similarly,information about 1 mm 500 µ m b f Spleen With tumors (actin Abstaining) g Smooth muscle and as 3D view in 25 d e f

µ m g c – Fig. 7a Fig. i ), ), 200 With tumors3D g h . The mouse lungs and mammary glands were µ – m. m. ( f Merge ) and lymph nodesand ) lymph ( i h g 200 j – l µ ) Optical ) projections Optical from the m d h Lymph node l j k 3D (crossview) With tumors 3D (crossview) 100 l k j µ Merge m Fig. 7g Fig. – l ). ). - © 2012 Nature America, Inc. All rights reserved. 8. 7. 6. 5. 4. 3. 2. 1. com/re at online available is information permissions and Reprints Published online at interests (see the HTML version of this article for details). CO All authors edited the paper. H.-U.D. led the development of the ultramicroscopy and clearing technology. contributed to the development of the clearing protocol at various stages. various clearing protocols. A.E., K.B., N.J., C.P.M., F.H., F.B., M.S. and H.-U.D. and J.G.E. provided transgenic mice. K.B. and N.J. developed and performed and videos. A.E. and M.S. wrote the paper. C.D.H. prepared the immune organs performed all the experiments, analyzed the data, and made all the figures AUT the Theodor Körner Fonds. supported by Genentech and the Hertie foundation. N. Jährling was supported by C. Sakanaka (Genentech) for providing mammary gland tissues. The work is GFP mice. We thank C. Murriel (Genentech) for providing the lung tissues and CD11c YFP-Venus mice, and D.R. Littman (New York University) for CX3CR1- Technology) for MHCII-GFP mice, M. Nussenzweig (Rockefeller University) for of Technology) for Kras in St. Louis) for the Thy-1 GFP (GFP-M) line, T. Jacks (Massachusetts Institute scale imaging microscopy and macros. We thank J. Sanes (Washington University manuscript, and C. Chalouni, H. Ngu and L. Komuves for assistance with large- A is availableNote: information in the Supplementary ckno

M H PET 135–143 (2000). 135–143 scaling. axial and shrinking Tissue I. images: confocal three-dimensionalreconstructionsfrom for methods Correction Xi Fen Pu clearing]. optical skin human on agents clearing specimens. Präparaten tierischen clearing. tomography.optical Diagnosis Medical system. nervous central injured the in axons spared fromregenerated distinguishing Bucher, D., Scholz, M., Stetter, M., Obermayer, K. & Pfluger, H.J. & Obermayer,K. M., Stetter, M., Scholz, Bucher,D., Q.L. Zhao, cleared or living of microscopy sheet Light H.U. Dodt, Keller,P.J.& W.Spalteholz, immersion V.V.optical Tuchin, Tissue & Bashkatov,A.N. E.A., Genina, diffuse for algorithms linearized of limitationfundamental A D. Boas, V.Tuchin, resurrections: False Tessier-Lavigne,M. & B. Zheng,O., Steward, OR w prints/index.htm I

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Expert Rev. Med. Devices Med. Rev. Expert NANC 31 Tissue Optics: Light Scattering Methods and Instruments for Instruments and Methods Scattering Light Optics: Tissue I et al. et ents Curr. Opin. Neurobiol. Opin. Curr. BUT , 302–307 (2011). 302–307 , Über das Durchsichtigmachen von menschlichen und menschlichen von Durchsichtigmachen das Über http://www.nature.c I I

[ AL J. Comp. Neurol. Comp. J. We thank M. Chang and F. Yeh for critically reading the ONS (SPIE Press, 2007). Press, (SPIE LSLG12D In vivo In l I . (S. Hierzel, 1914). Hierzel, (S. Opt. Express Opt. NTERESTS

A.E. A.E. initiated the current project, designed and mice, H. Ploegh (Massachusetts Institute of reflectance spectroscopy study of different of study spectroscopy reflectance

The authors declare competing financial

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