EIEllNttERNAttiONA[

Gold Coniugates

Technical lnformation and Protocols for use in:

0)Electron Microscopy

● Light Microscopy

C)Inllnunoblotting 「

Contents

1. lntroduction

2. Technical Assistancc

3. Spccimen Preparation 3.l Fixation 3.2 Washing 3.3 Ernbedding 3.4 Sectionlng 3.5 Etching 3.5.l Methods of Etching

4. lncubadons 4.l Choicc of Gold Coniugate 4.2 1ncubation BufFer(Buffer A) 4.3 Dilutions 4.4 1ncubation Procedure

(i) Blochng (il) P五 mary antibody (面 ) WaShing (iV) SCCOndary antibody (V) WaShing (Vi) SilVer Enhancing (Vii) COunterstdning Specinc lnlmun。 labclling Protocols 5.l Post― embedding labelling tissucs for EM 5.2 Pre― embedding labelling issues for EM 5.3 Pre― embcdding labelling of cell surfacc antigens for EM 5.4 Double labelling of EM sections

(1) Labclling both sides of the section (五 ) Labelling the same side of the section 5.5 1nlmunolabelling following in situ hyb五 disation for EM 5.6 1mmunolabelling of paranhn embedded scctions for LM 5.7 Prc― embedding labclling of tissues for LM 5.8 Labelling of cell surface antigens for LM 5.9 1mmunolabelling following in situ hyb五 disation for LM 6. Trouble shooting(nlicrOSCOpy) 61 Poor labclling 6.2 Excessive background labelling 6.3 Clustc五 ng 64 Buffcr recipcs 7. IIIImunoblotting of nucleic acids and proteins(BL Grade) 7.l lmmunostaining of DNA gel transfers(Southem blots) 7.2 1nlmunostaining of protein gel transfers(Wcstem blots) 7.3 Trouble shooting(membranes)

(i) Poo■ labelling (ii) Excessive background 8. Controls

9. Bibliography Introduction 7.2 1m】 』 BBI Gold CottugatCS arc madc to thc highest possible speciflcation and will givc thc 四 bcst possible rcsults in all applications when corectly uscd. This bookletis designed to ） help achicvc the best results by giving attcntion to specimen prcparation,choicc of gold COttugate and incubation conditions with diffcrcnt specimen types lt is not comprchcnsivc. Many books and articlcs havc bcen 、vHtten dcscHbing thc cxtcnsiVC (b) Tral pro( tcchniqucs ofimmunogold labcning This booklet、 vill,ho、 vcvct Cndeavourto desc五 be the ftlndamental factors 、vhich influencc thc rcsult that can bc obtaincd, suggcst a ＞ ｃ Was number of commonly uscd protocols,and pointto s01utions to common problems. の Blo( 2. Technical Assistance (C) Trar ln addition to the infomation givcn hcrc,our Tcchnical Scrviccs IDepartrncnt is al、 vays lnCL ready to givc hclp, guidancc, analysis and trouble shooting suggcstions to a1l our VaS customers. Please feel welcome to contact us directly or through our world― widc (0 ｀ dist五 butors for discussion of your applications Much of the infomation givcn hcrc is amplined in the introductolly Chaptcrs of our Rcscarch Products Cataloguc I)lease (g) ImΠ agit` request a copy Of our Cataloguc from your local distributor or dircctly from BBI. houl

3. Specilnen Preparation (h) 恥ras 、vat( Correct specilnen preparation proccdurcs frc absolutely crucial for optimal labcning of Gol( antigcns in cells and tissucs Mcthods commonly uscd for ultrastructural preservation COllJ in the EM or LM Inust usualy be modiicd to cnsure that antigcns arc not only rctaincd but also acccssiblc for labening.This often involves comprollllse、 vith the structure but (1) Imll carcful selection of preparation methods can yicld excellent combinations of stmctural nun detail and immunochenlical labclling Scveral dilfcrcnt spccilnen preparation methods as bl havc bccn dcscribcd in thc literature and arc comprchcnsivcly covcrcd in thc tcxts listed at the cnd of this booklct.It、vould not bc possible to suminanse an ofthem in 7.3 コЮl this sholttcxt Howcvcrthc fundamental stcps for each mcthod arc dcscribcd hcrc and protocols、 vhich fo‖ o、 v indicatc silnplc mcthods for post embedding labening as well All of the as prccmbedding labclling tcchniques with tissues as wcll as a simple approach to incubationl labening Celi sudace antigens in cen suspcnsions. Pool

3.l Fixation ａ ＞ Chc Cells and tissues may be nxed for subscqucnt cxanlination or may bc somctinlcs Ｄ High labcllcd in thc unixcd statc.Fixativcs either denaturc tissuc proteins by coagulation(eg Ｄ acctone or rnethanol)or by fOrming additivc cross linking compounds(Cg aldchydcs), Exc( or both.Thc rcsulting complcxcs incvitably dilfer from thc unixed protein in both their chclnical and antigenic pro■ les Each tissuc rcquires its o、 vn flxation protocol. For (a) Inadl cxample,too much cross linttng in a tissue、 vith high protcin density may mask many Saturate fo antigens On thc othcr hand,a loosc tiSSuC、 vith low protein contcnt rnay disintcgratc the gold cl without adequate fixation and antigens may simply be、 vashed out sulphurヽ V For many tissues thc best compromise is a mixture offorlnaldchyde(eg 2-4%)for rapid stabilising with low cross linkng,and weak glutaraldehyde(eg 01%)fOr greater structural preser17ation.For cytological invcstigation a prccipitating or coagulating flx such as acetone or rnethanol rnay be preferred Forlnal acetone has also been uscd for rlxing Cen preparations ln some studics simple air drying may a1low cnough antigenic prcservation for immunolabcning.

Post flxation for electron・ microscopy has lnostly involved the usc of osnlium tetroxide in order to prescrve mcmbranc componcnts and providc image contrast. Thc introduction of osnuum into tissue is,howevet not always desirablc and morc rcccntly tannic acid has bccn suggcstcd as an altemativc(1)。 Whatever mcthod of ixation is selected it must serve the dual function of retaining thc cssential strtlctural and antigenic components of the tissuc 、vithout introducing any matenal which may interfcrc、 vith the labelling. In somc cases thc introduction of hcavy mctals such as osnuunl or uranium into the tissue may causc increased non spccific labclling and must be trcatcd with caution.

Rcference:

1.M A Bettman etal(1992)“ Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochenustry"J Histochem Cytochcm 40,6,845- 857

3.2 Washing

Thorough washing of thc tissuc following ixation is extremcly important.It may sometimes be necessary to、 vash for atlcast as long as thc tissuc has been ixed in order to rcmovc cxCeSS aldehydc or othcr flxation rcsidues which may causc non speciic labelling ln some cases quenching thc tissuc with ammoniunl chloHdc is pcrforlncd to neutralise aldehyde groups.The wash is best perforlncd in a buffer having a tonicity

ゞ1雨ヽ冨ω the n血1■ ヽtss氏 .

3.3 Embedding

Tissues rnay be embeddcd in dimcrcnttypCs ofresin for EM applications.In cither case thc embedding should a1low good preservation of the antigens of thc antigens withOut s¨nkcグ stuctu減 ジ″物塑″θ嘱3勢領ぷy肋∝are l″ ο、ぃ ″SJtt cP"rCSins (cg Araldite,Epon)whiCh have an aromatic ring structure and arc strongly “cross linkcd, and acrylic rcsins(cg UNICRYL)which have a lowcr cross linklng.Epoxy resins are hydrophobic whereas acrylic resins may bc hydrophobic or hydrophilic Thc bcst structural prcscrvaliOn and stability is provided by cpoxy rcsins while thc best irrlnlunolabelling is achieved with acryliC resins.「 Fhis is because acrylic resins, espccially l」NI(〕 RYL,cutin such a、 vay as to rcveal the proteins at thc scction surfacc and they also wetrnore easilン ちthus giving greater acccssibility to the antibodies duHng subscqucnt incubations. For optimal rcsults a compronlise must be reached and the usual choice is a polar(iC hydrophilic)reSin with moderate cross linhng.UNICRYL, manufacturcd by BBI,gives excellcnt immunolabelling characteristics together with a 6.4 Bu田 high degrec of stability and structural preservation.Lrrゞ ICRYL allows thc cmbedding prOccdurc to bc perfomcd atlow tcmpcraturcs.This cnsures that vital componcnts are Thc baslc not extracted duHng dchydration and that no exccssive tcmpcrature nsc occurs during concentratl polymerisation which may damage the tissue antigens. (a) TnS For LM applications tissucs lnay be embcddcd in acrylic resins such as UNICRYL as described abovc, or in paraffin wax Resin cmbedded scctions providc very high Add 100m rcsolution of labelling but only a1lo、 v labclling of thOsc proteins at thc suFace Wax +0.242g(1 cmbcddcd sections pcrmit thc v/holc dcpth of thc section to bc labelled whcn +0.13g(21 de、vaxed Whcre high temperaturcs must bc avoided cryoscctions can somctimcs be +09g (22 used for ilninunolabclling for LM studics. Attust tO 3.4 Sectioning llllCrOflltcr

Scctions should be mounted on nickel or gold grids for EM invcstigations. Copper (b) PhOS grids introduce unwantcd background into thc immunolabclling For post embcdding labclling scction thickncss is unimportant since thc antibodies do not pcnetrate the Add 100m rcsin scctions.Scctions of 100nlll thickness should be adcquatc.While epoxy rcsin +0148gヽ scctions float easily and flattcn onto thc surfacc of、 vatcr,acwlic resin scctions require +0 043g К a more careful approach.Flattening is best achicvcd by cutting atthe nght spccd with +072g N〔 sharp knivcs,diamond if possiblc Thcy may bc mountcd on Formvar coatcd gnds or +0 13g Nで barc grids.The uscr should bc a、 varc that Fomvar and epoxy rcsin contains high levcls of sulphur、 vhich produces non spccinc attraction of gold particles AttusttO r

lt is also important to rcmcmbcr that itis thc proteins at the section surfacc which will 7. InIII bc cxposcd to subsequent solutions for labclling and that intcrnal protein、 vill not bc acccssed in rcsin scctions ln this respect it should also bc noted that thc protcins on Blotting ar thc scction surface 、vill be exposed to 、vater, not buffcL during the sectioning antibodies procedure and that this may affcct thc subscquent labclling to somc dcgrcc. This gene scqu( exposurc should bc kcpt as short as possiblc to avoid denatuHng thc proteins For thcse protc similar rcasons sections cut fronl rcsin embeddcd tissuc will not necessaHly havc an and South( ininitc lifc tilne、 vhen storcd at ambicnt conditions Frcshly cut scctiOns arc likely to be more rcliablc.To avoid leaching of proteins in、 vater somc investigators havc cut For Southc scctions onto buffer immcdiatcly bcforc incubating thcnl、 vith antibodics. produced t transfcrrcd If a tissuc block is sectioncd then itrnay oftcn bc obseⅣ cd that sections from the outer DN力RN´ region of the block will havc bctter structural intcgnty and lowcr labelling intcnsity biotinylate than scctions from thc centre of the block This is relatcd to thc penetration of thc flxativc and the degree of cross linkng and antigen rctcntiono When compa五 ng results Forヽ石estcr between incubations always usc attaCent tissue scctions blots of pr( of it,is inc For LM sections cmbcddcd in rcsin thc abovc considcrations are also truc.For、 vax cmbcddcd scctions,ho、 vever,the thicker the section the greatcr thc labclling intensity ln all cascs after de、vaxing. マVith LM scctions mounting securcly on glass slidcs is of grcat thc illllnob importancc to 、vithstand furthcr incubations Slidcs should bc irst coatcd with probc The

―

ピ B10BOND(from BBI)tO eliminatc vanations in glass composition from different sources and to provide a irln scction adhesive without introducing non― speciflc background.Lysinc should be avoided at an costs as a tissue adhesive since it attracts gold vcry strongly Chrome alunl also produces signiicant background

3.5 Etching lfthc immunoreactivity is weak or non existentsomc attcmptrnay be made to unmask antigcns fronl resin cmbcddcd scctions for LM or EM in order to makc thcnl morc accessible to thc antibodics.This maskng of antibodies may be duc to cross linHng by thc aldchydc ixation,chenlical intcraction with thc rcsin,covcring with other cross linked protcins,osnucation,covcring Of epitopes with resin,etc When using ctching techniqucs it is important to avoid causing furthcr damagc to thc cpitopcs 、vhile optilnally removing thc rcsin or digesting the surounding protcins ln general etching is not adviscd bccause of the dificulty of controlling the reaction.For ac,liC resins it is almost totally ineffectivc anyway.

3.5。 l ⅣIethods of etching

(a)For scctions oftissuc post ixed with osmium tctroxidc,trcat scctions with l-4% hydrogen peroxide solution for 5-10 min at room tcmpcrature This is an oxidising reaction 、vhich removcs rcduced osmium metal and makcs thc scctions morc hydrophilic Wash scctions thoroughly in 、vatcr bcforc pcrforlning noHmal incubations.

(b) Treat sections of osmium post ixed tissuc with saturated sodium mctapc壷 odate (0.1%dilutcd in distilled watcr)fOr 3-5 min at room tcmpcrature.This acts in a similar way to hydrogen pcroxidc(a).

(C) TrCat epoxy sections、 vith a saturated solution of sodiunl hydroxidc in cthanol or methanol for 30 1nin at roo■ l tcmpcraturc. This removes epoxy rcsin from sections.Difflcult to control for EM scctions.

(d) Treat ac■ /1ic sections with 70-100% ethanol This partially dissolves the methac,late bOnds and rcmoves resin slov/1y.Acrylics feel soapy v′ hen trcatcd with alcohol for this rcason

(e)TrCat acrylic rcsins with weak hydrochloHc acid(0.01-0.1%)fOr lo ndn(on gold g五 ds only)ThiS gradually removes the resin by interfe五 ng with the cstcr bonds E)ifficult to control without affccting antigenicity.

(O Treat sections with O.1%trypsin in TBS for 30 11un at 37C This unmasks antigens from unrcactive prccursors and digests surrounding epitopcs Uscful for trcating heavily Axcd tissuc(eg aftcr glutaraldchyde).Difficult to control selcctive digestion without intefcring with spcciflc cpitopcs. 4. Incubations (e, ヽb 岬 {1, 4。 l Choice of Gold ConJugate は Cold coniugates may be used directly or indircctly to label antigens in cells and tissues. い In the dircct mcthod the gold is cottugated to the p五 mary antibodゝ whether monoclonal or polyclonal. This a1lows a single incubation to be perfomed and provides thc simplest dctcctiOn system. In the indirect method a prirnary unlabelled 資 antibody is applied to the specilnen to locate the antigen.This is fonowed by a gold い labelled sccondary antibody that detects thc primary antibody This gold labelled ご1ご 軋エ secondary antibody is almost always an aFlnity pu壺 fied polyclonal. In this way an 1〔 咀 ampliflcation of the signal is achieved, often up to 10x compared with a direct incubation,according to the particle size.It fOr example,the pHmary andbody is from ゝL｀ mousc(a monOC10nal)thCn the second gold labelled antibody will be Goat anti― mouse. 選 A full range of BBI secondary gold labelled antibodies is given, togcther with full 「 guidelines for rnkng the correct choicc,in the BBI Cataloguc,Chapter 3 The indirect Cm mcthod for inlmunolabening is the most conlmon for studying antigens in cells and tissucs and avoids the need to label cvery pHrnary antibody. It providcs a universal 1. b」 ヽ method for detecting any pnmary antibody from the same p五 rnary species,ic all rabbit こ・ミ antibodies may be detected by Goat anti― rabbit gold cottugatCS,etc.The indirect method is longer than te direct rnethod because of the need for two or three scparate F, incubations butis the more widely uscd.The protocols desc五 bed below use the indirect “ method exclusivcly. IClE31

The choice of gold particlc size depends upon the magnification which is to be used in 平 the EM.In p五 nciple smaner gold particles produce a highcr labclling intcnsity due to their greater concentration in solution and the lower ste五 c hindrance produced. For lb, 国 example an lgG may be appЮ ximately 8nm in length.Gold particles of lnm or 5nm produce very little ste壺 c hindrance to the antibody activity9 while larger particle sizes ヽ∞ reducc the potential labelling intensity due to their sheer size. Larger pmicle sizes, tth記 howevet are mcDrc easily seen at lower magniflcations. For those users undecidcd about which particle size to choose for preliminary investigations, 10nm gold {d) Hヽ d COniugatcs are prcferred since these may be clcarly seen over a wide range of 2〔 )ご magnifications in the EM. ヽcll

leヽ e For ultra high sensiti宙 ty the vcry smallest,lnm,gold cottugatcs may be prcfcrred to f` since the number of gold particles attaching tO the tissue will be very high.This 呻 requres that extensive washing following the incubations is necessary.These particles ヽ are invisiblc,howcvet at magnifications below 500,000x and win need to be silver 胴 enhanced. Silver enhancing allows the particles to grow to any size suitablc, the 伽・ reaction stopped simply by washing in watei Silver enhancing procedures for EM 』 applications are descHbed below and are identical to those for LM use.

For LM studies,where silver enhancing is required,the choice is between lnln and {h)磯 5nm gold conJugates.lnm conJugates provide very lntense labening and can penetrate … ceⅡ membrancs duHng precmbedding routines Howcver very thorough washing is rcquired to prcvcnt un、 vanted background foHowing the incubations.

4.2 1ncubation Buffer(BuIFer A)

A typical incubating bufFcr suitable for both EM and LM incubations,which provides the componcnts for elinlinating the sources of background may bc as follows:

PBS or TBS +1%Norlnal serum(cg gOat scrLlm if using Goat anti― rabbit lgG:gold) +01%Tween 20 +1%BSA +0 1%sodium azidc attuSted tO pH8.2.

This is referred to as bufFer A in the folowing tcxt and is typical ofthc bu∬ cr rcquired for each step of an incubation.

The PBS or TBS providcs thc tonicity for antibody reaction.Antibodies win not work in thc abscnce of adequate salt.

The nomal scrum covers tissue sitcs、vhich may othcrwise attract thc gold labcllcd antibodics non speciflcany

The BSA blocks other``sticky''tissue components which would otherwise attract the antibodies non specincally.

Thc Twccn 20 rcduccs thc possibility of hydrophobic attraction bct、 veen tissue componcnts and the gold pttnicles

The pH of8.2 is often prefcrrcd to rcducc background caused by charge attraction of gold particles to positivcly chargcd tissue components At highcr pH valucs there is less positivc chlsc on proteins in the tissuc and thus lcss attraction for negatively chaBcd gold particles This is usuany the main reason for non speciic background and is cured silnply by raising the pH of thc incubating buffer ln order to achicve the highest possible signal with the lowest possible non speciic background it is important to be aware of an thc factors in both specimcn preparation and in subsequcntincubations which can arcct these results.For the indirect,two stcp mcthod ofimmunolabelling attention must be givcn to thc quality ofthe antibodics,thc nccessary dilution of each onc,the buffers uscd,and the incubation protocol.Wherever possible the method should be kept as simple as possiblc and each step or component must bc questioned as to its value.Thcre is no point adding rnany ingrcdicnts to a buffcr in order to avoid background if no such background c対 sts.It may actually cause the introduction of unwanted background! 4。 3 Dilutions (c) Fix 66 When establishing a new protocol it is necessary to deterlnine the optimum concentrations of both the p五 Fnary and the secondary gold labelled antibodes.This is (0 Rir achieved by irstincubating separate sections with v前 ous dilutions ofthe pHFnary Over an appropriatc range(eg 1/100- 1/10,000).The VariOus pHrnary incubations arc then (3) Inc followed by second incubations,using a constant dilution,such as 1/100,of the gold sen labclled second antibody.The dilution of the p五 rnary antibOdy which gives thc best signa1/background ratio is thus deterlmned.The procedure is then repcatcd,this time (h)Rn using thc chosen p五 rnary antibody dilution and varying thc gold labelled secondary antibody dilution ovcr arangc ot say9 1/10 -1/200.In this way the best dilution ofboth (1) POS antibodies will be deterrmned G)Wa 4。 4 1ncubation procedure (k)Silヽ The nomal steps to be taken in incubation protocols are as follows: Rin (1) B10C鳳 ng(where necessary) (11) PHmary antibody (m)COt (1li) Washing (iV) SecOndary antibody(gold labelled) (n) MOl (V) WaShing (Vi)SilVCr cnhancing(Where required) (0)Ⅵ el (Vii)COunterstttning(whcrC rcquired) Reference

These are discussed in general below and in the speciflc protocols、 vhich follo、 1 ～ l.M dcヽ Blocking smears wl

Non speciic reacuve sites on tissues and cell surfaces may nced blockng before 2.M DeV antibodies are applied to the specimens. This can reducc potential non― spcciflc nucroscop attrac● on of the pHFntt Or sccondary antibodies to thc tissue componcnts or the resin Brussels. material.Blocking reagents arc oftcn uscd bcfore the p五 rnary antibody step and can bc added to tte bumerin each step also.They includc: 5。 9 1mm

(a) BSA to cover non speciflc“ sticky"sites of tissue that would odlerwise attract Methods f proteins in gcneral.BSA also masks aldehyde groups which may still be present sectlons al in the tissue.Typically BSA blocking uses solutions of up to 10%.Thc BSA them.■ e solution should be freshly made and microttltered to avoid clumps of protein prchybndi attaching to the section and producing unwanted visual contamination. digoxigeni employed (b)Norrnal serum ofthe second antibody species(eg nOrmal goatserum when using lf digoxig( Goat anti―rabbit gold cottugates).ThiS Overcomes the possible atttac● on of are emplol tissue components for goat antibodies per se.Norlnal(ie nOn immune)serum should also be nucroflltered before use and should be heatinactivated.It is often (a) Perf used up to 5%in blocMng procedurcs.Unfortunatcly nomal goat serum is with relatively impure and can add un、 vantcd impu五 tics to thc incubation bulfe■ It should only be used if ncccssary Thc Cross rcactivity bct、 vccn thc nomal sertlm and the pHrntt or sCCOndtt antibOdies may not always bc well dcflncd.

As 、vith all othcr components in the incubation procedure,blocttng rcagcnts should only bc uscd if nccessary.(Dthcr、 visc thcy may introducc unknown signals into thc inal illllmunolabclling.Itis bestto avoid using blocに ng proccdurcs until itis found neccssary to dO SO・

(li) Prilnary antibody

Thc primaw antibody should be ofthc highesttitre and ofthe highest spcciic punty to especially、 vith thc allo、v thc highcst possible dilution Cross rcactivity must bc lo、 ～ち samplc tissue A lo、v quality p● rnary antibody is the greatcst causc of lo、 v specinc signal and high background du五 ng the labclling proccdure The bufFcr composition and the pH is important forthe incubations A norlllal TBS or PBS bumeris usually chosen 、vith suitablc additions to maintain a lo、 v background as desc五 bed abovc.Bu“cr A is typical The causcs of non speciic background are desc五 bcd bclow with their remcdics The dilution ofthc pHrntt antibody is discussed above The spccimcn must bc washed thoroughly with buffcr bct、vecn incubations

(ili) Washing

The tissuc scction is thoroughly washed in thc samc buffcr uscd(cg buffcr A)to dilute the p五 rnary antibody. Thorough 、vashing is nccdcd to rcmove ali non spcciically attached p五 rnary antibodies.

(iV) SecOndaw antibody

A high quality sccondary antibody is esscntialto labclthc p五 rnary speciflcally and、 vith low background BBI gold conJugatcs are arlnity punfled and of thc highest quality Thc cottugatCS COntain no frcc antibody all antibodics bcing adsorbed sccurcly to the surfacc of the gold particles.BBI gold co可 ugateS need not and should not be centrifuged.Their high titrc and punty mcans that for incubation they may bc highly dilutcd(typiCally 1/10-1/400)in thC typical incubating burcr shown abovc(ie samC as for thc p五 rnaw antibody),sO reducing non― spcciic background whilst achicving a high signal intensity

(V) WaShing

Thc tissue section must again be thoroughly v′ ashed、vith thc samc bu“er to removc un、vantcd non― spcciic gold patticles.Aftcr washing in butter the scction must also be thoroughly washcd in distined 、vatcr to rcmovc all traccs of salt and loose protcin bcfore it is allowcd to dry.

Du五 ng the incubations,a grcatcr sensitivity may bc achieved by constantly flushing or agitating thc tissue section in the solutions.This may bc achicved on a gcntlc shaker platforln or by irrllnersing the EM section in a small tube、 vith the incubating solutions Cataloguc and Placing it on a rollcr bed, so bHnging frcsh solution continuously to the tissue labclling: su」∝e proteins as would be the casc in vivo.If thc antibody solutions are tolerant the biotin or incubations may also be perforlned at 37C to nullllc in ViVo conditions further havc bcen detection lf floating sections on the surface of droplets of andbody,it is important to prcvent any con」 ugatcl antibody solutions from drying on eithcr side of the dssue section.Drying can produce non speciflc aggregations of gold particlcs across thc sec●on surface (a) PC責 inis (宙 ) SilVer Enhancing sect

Silver enhancing may bc uscd for EM studies where thc gold paniclcs arc too sman to (b) Plac be seen in the available rrllcroscope or to grow thenl fronl any o五 ginal size to a size that norr givcs a more comprehcnsivc view of labelling at lower inagniflcations The inethod is use quite easy to pcrforlll for any onginal gold particlc sizc choscn.In ol・dcr to cnsurc that strcl the gold labcls arc not rcmoved froln thc section it is collllnOn to ix thc section for 10 ■linutcs in l,ろ glutaraldchyde in watcr bcfore silvcr cnhancing.Using the BBI Silver Enhancing Kit for LM alld EM (SEKL)a drop Of Cnhanccr and initiator are血 xed (C)Trar togcther on a clean petn dish and thc gold labelled EM scction placcd on thc surfacc or for i evcn immersed in the dЮ plet.Thc time ofenhancement must bcjudgcd cmpincally but approxinlately 5-10 rninutesis usuany sufEcientto grow fЮ m lnm to 5nm,or fron1 5nm (d)Trar to 20nm.It is temperature depcndcnt.The enhancemcnt proccss iS StOppcd by washing mon in distilled、 vater and thc g五 ds rnay bc re― enhanced aftcr cxallllning in thc EM until thc choscn sizc is achicvcd Provided the sections are not exposed to an electron bcam, Optional s silver cnhanccd scctions may be furthcr inllnunolabelled in a subsequent incubation proccdure This may provide another rncthod of double labelling. (C) If rc (SE】 For LM studies silver enhancing is a necessary but straightfonvard proccdure cxpc

(宙 i)COuntersmining (O WaS

Scctions whch have been immunogold labelled inay also bc countcrstaincd by the usual (g) COu: mcthods of uranyl acetatc and lcad citrate Again,the use of glutarddehyde will reduce thc loss of gold particles from thc scction surface. 5.` 11■n

5。 SpeciFIc lnIInunolabelling PHDtOC01S (a) Dew The following protocols are provided as a guide for irrlmunolabelling different tissue and cell samplcs in thc EM and LM. Many others exist and may be found in the (b) Equi literature.These protocols scrvc to indicate gcneral approaches to achieving maximum sensitivity and nllnimum background. (C) Plac gold

5.l Post embedding labelling tissues for】 ]Ⅳl scrul or Pl (a) Placc the g五 d,scction downwards,on a 50ul droplet of 10%heat inactivated nomal serum(of thC gold labelled animal spccies,cg for Goat anti― rabbit:gold (d)Witr use nonmal goat scrunl)in TBs or PBS bu∬ er and incubate for 10 min.For antil PЮtein A,Protcin G or Protein A/G gold cottugates Omit this stcp a dis

10 rithout washing,gently blot olF excess and transfer the g五 (b) ヽ d to the surface of a 50ul droplet of p五mary antibody diluted in bu“ er A(above).InCubate for 30 nlln to ovemight.

Transfer thc g五 d to a se五 es of 5 or rnore 50ul droplets of burer A,lcaving 5 Hlln on each,to rcmove unbound antibody.

(d) Transfer thc g五 d to two suCCessive 50ul droplets of gold cottugate diluted in thc approp五 ate bumer A The list drop will remove cxcess bumer and the second drop will provide thc incubation.Incubate for l-4 hours.

Transfer thc g五 d to a sedes of 50ul droplets of distillCd Water(5x2minutcs or mOre)tO removc unbound gold coniugate.

Optional silver enhancement:

(O If rCquired place the gHd on a drop of freshly nllxed silver enhancing solution (SEKL)from BBI fOr a pe五 od of 5 - 10 minutes.Dctermne the time expe五rnentally beforehand by enhancing gold particles which have been allowed to dry on a Fomlvar coated g五 d.

(g) WaSh thOroughly in distilled watcr to stop thc rcaction.

(h) COunterstain if requLd with uranyl acctate and lead citrate as nomal.Frozen sec●ons rnay be stdned with oslnum tetroxide vapour Wash and examinc in the electron nllcroscopc. ５２ 。 I Pre‐embedding labelling tissues for I]Ⅳ ０ Dissect thc tissue to the smallest possible size (eg O.5mm)for handling and good penetration.

(b) Lighdy fix tissuc in 2-49ら parafonnaldehydc+0.1%glutaraldehyde for 30-60 min depcnding on tissue size.

(C) WaSh tiSsue very thoroughly in PBS(eg 30-60 nin)tO removc cxCeSS aldchyde groups.

(d) OptiOnally pcmeabilise tissuc with O.1%Tween 20 or Tdton― X100 for 15-30 nun

ｅ ， Wash tissue very thoroughly in buffer A for l houn

０ lncubate tissue with 20 x volume of p五 rnary antibody,diluted in burer A,for l-4 hours while agitating. (g)WaSh in bu“ cr A vcry thoroughly,ie for l hour while agitating,to remove (h) InCl cxcess pHrnary antibody. 1/11

(h) InCubate tissue with gold coniugate suitably diluted in bufFer A for l‐ 4 hours (i)Wa while agittting.For most applications only lnm gold cottugates will penetrate the ccll membranes.lnm gold cottugatCS are usually dilutcd to 1/100-1/400 for G) Fix incubations. (O Wa (1) WaSh tiSsue very thoroughly in buffer A (30n」 n)and then in PBS for 30 n五 n. while agitating to removc excess gold cottugate. Optional: o) Fix tissue in l%glutaraldehyde for 10min to strengthen antibody binding to the (1) InCt tlssuc. det(

(k) WaSh thOroughly in water for 30 min while agitating to rcmove excess aldchyde and PBS (m)ヽ ヽ lons

Optional silver enhancing: (n) Spil LM (1) Inlmerse the tissue in fresh silvcr cnhancing solution(BBI SEKL)for 5-15 min. ExpeHment with a number of tissue blocks to deterlmne the time required. Reference ＜ ｍ Wash thoroughly in distilled water to stop enhancing. l.C Ferra ０ perforlnec Continue through to dehydration and resin embedding for EM or LM (frOm BB] lnvestlgatlons. ５ ３ ． Pre‐embedding labelling of cell surface antigens for EⅣ I 5.4 Dou ０ Isolatc cclls from tissue with trypsinisation or separate blood cells by gradient centnfugation. Spin to a soft penet and resuspcnd in subsequent incubation (1) Lab solutions. ln this me the lower (b) Prenx cclls in O.1% glutaraldehyde or 2-4% parafomaldehyde in PBS incubation depending on antigen sensitivity.

(C) WaSh 3 timesin PBS containing 50mM glycine to quench aldehydes. (a) Foll

(d) B10Ck Cells for 30 min in PBS+1%BSA+109ら nonnal senlln(seCOnd antibody (b) Opti species). (C) Tun (C) ｀ヽSh tWice with PBS+1%BSA. antil antil (O InCubate with p五 mary antibody diluted in buffer A for l hour(typically 1/10- seco 1/100 for a monoclonal) Bccause t、 (g) WaSh tWice in buffer A. antibodies

12 .to remove (h) Incubate in second gold labelled antibody diluted in burerAfor l hour(typically 1/10-1/100).

11-4 hours (1) waSh tWice in PBS ・im penctrate O-1/400 for G) Fix in l%glutaraldehyde in PBS for 15 min.

for 30血 n. (k) Wash tWiCc in distilled watc■ Optional silver enhancement: ulding to the ‐ (1) Incubatc in fresh silvcr cnhancing solution(BBI SEKL)for 5-10 min This is detcrlmned expcnmcntally

皓s aldehyde (no WaSh thOroughly in distilled wtterto stop thc enhancement and to remove silver ‐ ions from thc cells.

(n) Spin tO a flrrn pdlet,continue through to dehydration and embedding for EM or n as norlnal. or 5‐ 15 min. LM investigations.Counterst五

quれ d・ Rcference:

1.C Ferr触 ,ct al(1989)“ Preembedding inlmunogold st滅 ning of cell surface antigens 劃 or LM perfomed on suspended cells and dssue scc● ons''in Hayat MA,Colloidal Gold,Vo1 2 (frOm BBI).

5。 4 Double labening Of EⅣ I sections

hg boぬ ddesぱ ぬe sedon iZ認:認 ① Lab』 de h PBS l:u躙 tF就 狙 :l∫ 布f留躍:=1:kttiぷ観 おふ柵 艦留b:繁譜

s t (o Follow stcps 5.1(a― C)abOVe using the flrst pnmay antibody.

鴻d andbody ' (b)OptiOnally coat the reacted side of the section with carbon evaporation.

― (C) Tum the section over and repeat steps in 5.1(a― C)using the second pnmary antibody but also using a di“ ercnt gold p“ cle size for the second gold labelled antibody.Typically tts means using 5nm for the frst side and 15nm for thc pically 1/10- SecOnd side.

Because two different sides ofthe sccdon are incubatcd independendy血 3two pnmary antibodies may be raised in the same aninlal species.

13 Optional silver enhancement:

(d) If required either side of the scction may bc silver cnhanced.Place the g五 d on a drop of freshly lrllxed silver enhancing solution(SEKL)from BBI fOr a pe五 od of 5-10 minutes.Deterrmne the time expe五 rncntally.

Altemativelン Ъ the same particle size may bc used on both sides of the section and one side enhanced with silver to produce a direrent flnal sizc for double labening.

(C)WaSh thOroughly in distilled water to stop the reaction.

(O COunterstain ifrequired with uranyl acctate and lead citrate as nomal.Wash and exanune in the electron nucroscope.

(li)Labelling the same side of the section ln this rnethod both antigens are labellcd on the samc sidc of the section using difFerent particle sizes.The tissuc section may be mounted on a Forlnvar coated gHd for the incubations

(→ Follow steps 5.1(a― →abOve using the first pnmary antibody(eg frOm mouso.

(b) Repeat steps 5.1(a― e)abOVe but using a second p五 mary antibody from a diffcrcnt spccies(eg frOm rabbit)With the appropHate gold labelled antibody(eg Goat anti― rabbit).

Optional silver cnhanccment:

(C) The section may be silvcr enhanced after stcp(a)abOVe in order to grow the particles to any size required before perforlning the second incubation procedure. Follow stcps 5.1(f‐ g)Do not expose the section to the clectron beam beforc the second inlmunolabclling procedure sincc this will denature all proteins.

Counterstain:

(d) COuntcrstain if rcquired with uranyl acetate and lcad citrate as norlnal.Frozen sections rnay be stained with osnllum tetroxide vapour Wash and cxanunc in thc electron nucroscope.

5。 5 Lnmunolabening f0110wing in situ hybridisation for EⅣ l

Medlods for pcrfoming in situ hyb五 disation of nucleic acids in embedded EM tissue sections are、 videly reported in the litcrature and no attempt is made here to repeat them. Thc user is encouraged to read a good text book on in situ hybHdisation techniques before ventu五 ng into immunolabelling of target probes (see BBI

14 lting s01utions Cataloguc) The steps for in situ hybHdisation arc: spccilnen preparation; probe to the tlssuc labcning;prehybHdisation;hybridisation with a spcciic labellcd probc(labened with rc tolcrant thc biotin or digoxigenin), fOnOwed by antibody incubations. If biotin labened probcs inhct havc bccn cmployed thcn Goat anti― biotin or strepta宙 din gold cottugatcs are used for detcction. If digoxigcnin labcned probes are uscd then Sheep anti― digoxigenin g01d io prcvent any COttugates are employed. g can producc

(a) PCrfOrln the in situ hybHdisation steps accOrding to a suitablc protocol and flnish with st五 ngency washing procedurcs to remove unbound probe from the SCctlon.

re too small to ie to a sizc that (b) Placc thc gHd, section downwards,on a 50ul droplet Of 10%hcat inactivated Thc mcthOd is nomal scrum (of thC gold labcned animal species,cg fOr Goat anti― biotin:gold usc norlnal goat scrumi for Shccp anti― to ensurc that digoxigcnin usc nomal sheep serunl;for scction for 10 streptavidin use nothing)in「 FBS or PBS bufFcr and incubate for 10 inin. 山c BBI Silvcr itor are lnlxed (C) Transfcr the g五 d to a 50ul droplet ofgold cottugatc diluted in bumerA Incubate l he surfacc or for l-4 hours 31npincally but n.or fron■ 5nm (d) Transfer the ghd to a series of 50ul droplcts of distined watcr(5x2nlinutcs or cd by、vashing mOre)tO removc unbound gold cottugate. lc EⅣ I until the elcctron beanl, Optional silver enhancement: lent incubation (C) If rcquircd place the g五 d on a drop of freshly mixed silver enhancing solution (SEKL)from BBI fOr a pe五 od of 5 - 10 面 nutes Dctcrlninc thc timc 3dure. expcHrncntally.

(O WaSh thOroughly in distillcd water to stop the reaction lcd by thc usual (g) COunterstain if required with uranyl acctatc and lead citratc as normal. 、dc、vill rcducc

5。 6 島mmunolabelling of pararln embedded sections for LⅣ I

(a) Dcwax sections in xylcne and bring to watcr through eぬ yl alcohol dlffercnt tissue c found in the (b) Equilibratc thc tissuc scction in PBS for 30 1nin ving maxlmum (C) Placc On the scction a 50ul droplct of 10%heatinactivatcd nomal serum (of the gold labelled animal species, cg for Goat anti― rabbit:gold use nomal goat serum)in TBS Or PBS bufFcr and incubate for 30 min For Protcin A,PЮ tein G or Protein A/G gold conJugatCS Ollut this step leat inactlvatd mti― rabbitigold (d) WithOut washing, gcntly blot of cxccsS and replacc with 50ul of pHnlary Dr 10 min.For antibody dilutcd in bumer A(above).InCubatc for l hourto ovemight.Cover with ,p a dish to avoid evaporation.

15 ｅ ＞ ミ、sh the slide thoroughly by inlmersion in butter A for 30■ jn while agitaung.

０ Place on the section a 50ul droplet of gold cottugate(lnm or 5nm)diluted in buffer A.Incubate for l-4 hours.

(g) WaSh the slidc thoroughly by inlmersion in water for 15 Πin by agitation.

(h) Placc On the section 50ul of l%glutaraldehyde in water for 10 min to flx the COttugatc in place.Wash thoroughly in distilled water for 10 min by agitation.

(1) Place On thc section 50ul of freshly nllxed silver enhancing solution(SEIく L) from BBI for a pe五 od of 5- 10 ninutes.Detcrlmne the time expe五 mcntally du五 ng the enhancement procedure by observing in a light microscope.Do not expose to prolonged excessive light.

G) WaSh thOroughly in distillcd watr to stop the reaction.

(k) COunterstain using nomal histological stttns.

(1) MOuntthe section in BBI BIOMOUNT to preventfdng Ofthe signal produced by some other types of rnountant.

The silver enhancement stcp will convertthe invisible gold to first a brown stain sinular to a peroxidase stain and then to an intense black stain that will, under correct incubation procedures,be speciflc and localised with very good resolution.

5。 7 Pre…embedding labelling of tissues for LPI

(a) Follow the steps dcscHbed in 5.2(a‐ m)

(b) Dehydrate the tissuc and embed as usual for LM studies using either resin or parafrln wax.

(C) SeC●On and counterstain as requred.

5。 8 Labelling of ceH surface antigens for Lル I

(a) IS01江C Cells from● ssue with trypsinisation or separate blood cells by gradient cenmfugation.

(b) WaSh Cells in PBS+1%BSA and suspend t0 10 x 109 cells/1itre in PBS+5% BSA.

Prepare a cytocentrifuge preparation or make a smear from the butt coat OntO a glass slide cotted with BIOBOND.

(d) Air dry ovemight.

16 (e) Fix for 30-90 sccs with phosphate buttered 9.25%fonmol and 45%acetonc at pH 6.6. lc optlmum ldies This is (o Rinse thc slidc in PBS. 〕Πnlay Ovcr ons arc then (g) InCubate for 10 min in PBS+5%non― fat rrlllk or 0 01%casein+5%norlnal .of the gold serum(ofぬc g01d Cottugate)+required concentration ofthe gold cottugate. ヽcs thc best ごd.this time (h) Rinse with PBS. d secondary ュtion ofboth (i) POSt fiX the cells with bumered forlnal acetone for 2 min at room tcmperature

G) WaSh thC Slidcs thoroughly in distillcd wate■

(k) SilVer enhance whilc monito五 ng underthc LM.

(1) Rinse with distillcd water to stop enhancement.

(m)COunterstain with May―Grunwald― Giemsa stain

(n) MOuntin BIOMOUNT to prevent fading ofthe silver stdn.

(0) View in thc LM using b五 ght ield and epipolanscd light

Rcferences: お1low 1.M de Waele et al(1991)``Lcukaenna and lymphoma inllnunophenotyping in cell smears with inlmunogold silver staining''Am J Clin Path 96,3,351-359.

:klng before 2.M De Waele(1991)“ C0110idal gold as an immunocytochenucal maker for thc light non― speciflc 血croscope detection of leukocyte cell su」 acc antigens"PhD Thcsis, University of :or thc rcsin Brussels p and can be

5。 9 1mmunolabelling follo■ ving in situ hybridisation for LⅣ I m lse attract Mcthods for pcrforrrung in situ hybndisation of nucleic acids in embedded EM tissuc ll bc prescnt sections are widely reported in the literature and no attempt is madc here to repeat そThc BSA thcm.The steps for in situ hyb五 disation arc: specimcn preparation;probc labclling; )s of protein prehyb五 disation;hyb五 disation with a specific labclled probe(labcllcd with biotin or On digoxigenin),fol10WCd by antibody incubations. If biotin labelled probes have bccn employcd then Goat anti― biotin or strepta宙 din gold coniugates are uscd for dctcction. l Чhen uslng If digoxigcnin labcllcd probcs arc uscd thcn Sheep anti― digoxigenin gold cottugatCS attract10n of are employed. nune)SCrum 姐 It is oftcn (a) PerfOrln the in situ hyb五disation stcps according to a suitable protocol and inish )at serum ls with strlngency washing proccdurcs to rcmove unbound probc from the section.

17 ° 1籍樋朧l 驚攀轟轟難 (c) Rcplacc with a 50ul droplet of gold cottugate diluted in bu∬ cr A.Incubatc for l-4 hours.

(d) WaSh thc slide moroughly With distilled watcr for 30 min to remove unbound gold cottugate. ° 1きぶよ誰ゴII鉗:オ戦:lttt獄 棚F尾蹴:響l緊聴鵠導胞 groups. 『 °

甲 蝋 諸 椰 曹 鐵 椰 鵬 職 朧 鮮

(g) WaSh thOroughly in distilled watcr to stop the reactiOn.

(h) COuntcrstain with nomal histological stains.

(1) MOuntthc scction in BIOMollNT to avoid the fading ofthe silver enhancemcnt.

6. IIouble shooting(miCrclscopy)

棚 ‖::鍛::譜え:::lttI漑 lξ翼瓶品l憲枇翼:胤驚1犠鶉 h“ upon W鳳尋踏Hi響魔 1翼|::棚鍵Ⅷ∬ "n“ =調 ° 符風究再乱:庶 器:服竃:認器 僣滞 鳳:盟晰 五se too high du五 ng cmbedding=摺 or polymensation. (b) Andgcn prescnt in gcnuinely low alnounts. I」se longer incubatiOn times and ③more concentrated p五 rnary andbody. ∬絲鯛謬さぶrS稲 盤撃聾肌蠍∬覆i三』:神

③ 江et pЮttdngぬe hook e■d UК gК atcr 訛器需IPl月8,酬len廿

18 (C) WrOng g01d co巧 ugate.Chcck and rcpeat the incubation.

(O pH Of bufFcris wrong(eXCessive alkali or acid).CheCk S01utions ls and tissucs. (g) InSuf「lcicnt salt content in buffer to allow antibody intcractions. Salt content od、 whethcr should be atlcast 50mM 〕Jomcd and ぃ unlabclled (h) SCCtiOn not exposcd to solutions(ic wrOng way up)if On a plastic ilm. ted by a gold gold labclled (i) Labclling not visible due to heavy stains (Cg fOr 5nm particles). Rcduce counterstaining.Usc higher inagniflcation(Cg 200,000x for 5n■ 1, 100,000x for l this way an 10nm,80,000x for 15nm,50,000x for 20nln) 、ith a direct dbody is from sent)Use G) SilVer enhallcemcnt proccdure is incomplctc(stain is light bЮ wn orあ at antl― mousc. longer cnhancemcnttimcs Chcck positivc controls. ther with full ヽThc indirect (k) COuntcrst滅 ning is maskng he silvcr stdn Use lcss counterstain s in cclls and 3s a universal (1) SilVer stttn fades after solne timc Rcmovc covcrslip wim xylenc and wash in 〕s.ie all rabbit dcvelopc■ Remountin BIOMOW nc indirect thrcc scparate 6.2 Excessive backgrcDund labelling sc thc indircct (a) IOnic concentration too low in solutions Use incrcased salt concentration(up tO 2.5%)Add BSA or norlnal goat serLIm(nOt fOr Protein A,G,A/G)to sto be used in approximately 19ろ in incubation solutions. itcnsity due to Droduccd For (b) Inadequatc washing bctwcen incubations Thoroughly wash(hours if necessaγ ) f innl or 5nm particlc sizes (c) Non―speciic charge amaction of antibody Rdsc the pH ofthe burcrto pH8 2-8.5 particlc sizcs, to reducc thc posidvc chargc on tissue protclns ers undecldcd on of gold pmiclcstO ussue cOmponents.Increasc the Twcen ,. 10nln gold (d) HydrOphobic tttrac■ ,ldc rangc of 20 content to l-2%.

(c)Non spccinc attraction of sccond antibody to ussue components.Includc highcr levcls of norlnal serum(eg Goatserum for Go飩 anti― rabbitgold)in thC bumet up l be preferred to 5%. :D′ high.This

I司 hcsc particles (O hTuHdesin hc norma serum uscd Omit hc scrum and observc the rcsults 姐 to bc silvcr , suitablc, the (g) High levcls of sulphur attacung g。 ld particles in the tissue or cmbedding resin. dures for EM Change to ac,lic resins or includc up to O.01%gelatin in the gold cottugtte fOr mat step Only recn lnm and (h) Free aldehydc groups in dlc issuc. Reduce by ■oaung sections on 0 5M i can penctrate anlmoniunl chlo五 dc for l hour beforc incubations.

19 Primtt andbOdy concentadon too high.Dllute by orders ofmag丘 tude.恥 crever possible use afflnity punfled andbodies. ① Gold coniugate cOncentadon too high.Dllute mttЮ ■ ① Inadequatcly flxed tissuc. Necrotic and darnaged cells will also stain non― speciflcally.LnpЮve nxation conditions and use smaller pieces of ussuc.check 」К positive control.

(1) OSmium tetro対 de flxation may introduce excess charge into tissuc.Omit osnuunl or wash thoroughly aftcr fixing。

(m) Excessive cxposurc to silver enhancing s01utions, especially for low signal. Reduce the developmenttime.

(n) Excessive exposure to light duHng silver enhancement. Reduce thc light intcnsity.

(0) SpOntaneous precipitation of silver duc to impuHties in glassware and the water Use ultraclean glassware and distilled/delonised watcr of high punty.

(p) SpOntaneous precipitation of silver onto endogenous lnetals in tissuc(eg Zn3 Fe, Cd,ctc).Carefully check the negrative controls.

(q) Non speciflc attraction of gold to lysine when used as a tissue binding agcnt on slides.Avoid the use oflysine and use BIOBOND instead

`3 Clustering Clustenng descHbed here should not be confused with ampliflcation produced with indirect labelling. In the ampliflcation process each p五 rnary antibody may have attached several gold labened secondary antibodies.This win lead tO sman groupS Of gold particles,perhaps 2-4,which surround single antigen targets in the tissue section

For LM Grade and BL Grade conJugates clusteHng is of less importance since the particles are not seen individually in the LM sections or on blotted membranes. Nevertheless excessivc cluste五 ng du五ng manufacture is sdctly avoided since this would otherwise lcad to long tcrln instability Excessive clusteHng inay sometimes be seen on EM sections for the following reasons.

(a) Clumped p五 mary antibody.Use fresh andsera,Micro■ lter if possible to remove bactcHa.

(b) COntamination ofthe gold coniugate.Caused by lysine or other protein additives, by thimerosd or mercaptoethanol.CottugatC Stored at extreme pH.Cottugatc overheated.Store at 4C in separate aliquots in he o五 ginal burer supplied until required for use.Do not freeze and thaw frequently.Do not place pipettes into a single vial frequcndy.

20 Fther宙 dl a Bttr Recipes E embedding `“ mttЮnents are ‐ bぉ PBS and mS burers are Jven here.These are supメ emented with Hgher ocurs duing concentratlons“ ofTween 20,NaCl,or BSA as required.

(a) TriS buffered saline llNICRYL as lde very high Add lCXld distilled water sraceo wax +0.242g(20mM)TriS(triStydroxymedlyl― aFmnOmethanc) 雌 ued when +0.13g(20mM)NaN3 0reserVative) sometimes be +0.9g(225mM)NaCl

Adiust tO pH 8.2(or other pH as necessary)with O.lN NaOH or O.1%HCl and micdlα

ns.Copper O)PhOSphate burered saline Ы“ embedding penetrate the Add 10md disdlled water c epoxy resin +0.148g Na2HP04 dions require +0.043g KH2P04 典t Speed with +0.72g NaCl ted grids or +0.13g NaN3 “∞ntdns high es. AdiusttO pH Wiu1 0.lN NaOH orO.lN HCl and microfllは

7. unoblo硫 hg ofnucleic adds and Proteins GL GndeD =e which will ― 血 will not be he proteins on Blotting applications usingBBIGoldConiugates includedle demonstrationofandgens, 雌 sectioning antibodies,proteins,macromolecules,total DNA/RNA,and DNA/RNA iagments and じdegree. This gene sequences transfered to an i― obiLsing matrix.The procedures for transfer of 〕proteins.For these protelns and nucleotide sequences fouow ale wen estabushed methods ofWestem Mrily have an and Southern blotting,or direct dot blotting. 薦 are lkely to 昇Юrs have cut For Soualem or Nomem Ыots,for example,indi宙 dualfranentS Of DNAor RNA,as ES. produced by resmc■ On enzyme digestion,are separated by gel dectrophoresis and men transfered to an inert support such as nylon or nitrocemulose.¶ ℃ i―obilsed hm the outer DNttA fragmcnts can then be analysed by in situ hybridisation using a suitable dling intensity biotinylated cDNA pЮbe. EaatiOn Of the Waring resuhs For Westem blotting,protein dispersions from olle or"o dimensional gels,or tom dot blots ofproteins,are inlmobilised onto a mtrocenulose sheet and the whole sheet,or part of it,ls incubated in the appropnate p―ary antibody ,me.For wax cning intensity h au cases,BBI GoldGttugateS Of lnlnor20nIInparuclediametcrareusedto宙 sualise 魅 is of great the i―oblsed materids following incubation with an appropnate pnmary antibody or 菫 coated with probe.The lnln gold conJugateS glve much higher labening mtenslty but must be suver

21 cnhanccd to be visible The 20nm gold particles ae dircctly visiblc On the mcmbrane Thc lnm cottugatc alsO κquires cxtcns市 c washing to remOve unbound particlcs from the membranc.

Because ofthc inexpensivc and highly concentated nature ofthc BL Gradc conJugatcs, 1肛gc volumes may be used at high dilutions SOuthem blots from DNA gcls are best inlmobiliscd on nylon based membrancs whcrc binding is more cnhcicnt.Protcins bind more e“ cctively onto nitrocellulose ヽヽrith nylon mclnbranes,ho、 veve■ it is necessary to block unoccupied sitcs rnorc cxtensively attcr blotting to avoid background staining

A simple protocol for staining blottcd protein or DNA is givcn bclow.Blottcd protcins, antibodies or antigens may bc cxanllned by thc simplc t、 vo step indircct labcning method.E)NA/RNA fragments,labcllcd、 vith biotinylated probcs may be dctccted by using a gold labcllcd antibody to biOtin

7。 l lnllnunostaining of DNA gel transfers(Southern blo")

(a) PCrfOrm a norlnal silvcr stain or wholc DNA stain on a narrow stl■ p Of gel or duplicatc gel to gaugc thc total DNA pattcm.Altcmatively usc GENOGOLD (frOm BBI)tO prO宙de a total nuclcic acid stain on blottcd membrancs

(b) Transfcrthc DNA/RTA pattcm to thc immobilising mat五 x by establishcd blotting procedurcs and perfornl hybridisation expcriments as appropriatc 、vith biotinylated probcs.

(C) WaSh the membranc in PBS.

(d) B10Ck me membranc with 10%BSA to ill unoccupicd sites.Incubate the mcmbranc in dilutcd gold cottugatc(cg Goat anti― biotin:gold for biotinylated probcs)for l-24 hours whlc agitating.WhOlc sheets may bc incubatcd in flat plastic bags.IIigh dilutiOns of antibody will givc 10、 ver non spcciic background. Dilutions may be made in TBS or PBS supplemcnted with additions to rcducc background(scc 4.2)

(C) WaSh the mcmbranc thoroughly in butter with scveral changcs to rcmovc unbound antibody.

Gold cottugates Of 20nm will bc宙 siblc as rcd bands Thc lnm cottugates will requirc silvcr cnhancing

(0 Wash the membranc vcry thOrOughly in distilled watcr to removc all non speciflc gold and salts.

(g) Inlmcrsc the membrane in fresh silvcr cnhancing solution(SilVCr Enhancing Кat for Blotting,SEKB fЮ m BBI)and agitatc for 10-20面 n Stop thc Кaction by washing in watcr beforc the membrane tums grey.Thc DNA bands willappeT as black bands.

22 7.2 1mmunostaining of protein gel transfers(Western blo“ )

三: こ1、 c thc (a) PCrfOrln a Coomassic bluc stain on a stHp of thc gcl to deteminc thc wholc icsigncd to protcin staining pattcm Altemativcly usc PROTOGOLD(from BBI)On a scpalatc blotted membranc to givc a total protcin stain lご c of qold lt is not tcin pattem to thc immobilising mat五 x by establishcd blotting 3 こヽtCnSlヽ c (b) Transfer the pЮ procedures lご dcscrlbc

ゝuこ gcst a (C) ｀｀4ash the mcmbranc thoЮ ughly in PBS うlcnls (d) B10Ck thc unoccupicd sitcs by immcrsing the membrane in 10%BSA for30 min.

(e) Transfer thc mcmbranc to thc p五 maw antibOdy diluted in bu“ cr A(scc 4.2).

1l is al、 a、 s Incubate for l hour whilc agitating to a1l our cr A whilc agitaing for 30 min i orld― 、idc (fl WaSh thC mcmbranc vcw thorOughly in bu“ :'.cn herc l、 (g) Illlmcrse thc lllcmbranc in gold labcllcd second antibody dilutcd in bumcr A、 vhilc こuc Plcasc agitating I)ilution of thc antibody should be deterlmncd as in 4 3.Incubatc for l ■BBI hour

(h) WaSh thC membrane vcw thoroughly in bumcr A for 30コ dn and thcn in distilled 、vatcr for 30 min、 vhile agitating

:こ ol Gold cottugatCS Of 20nm will appea宙 siblc dircctly as red stains.For lnm gold ,rc"llin■ sc、 こtlon COttugatcs silvcr cnhancing is nccessay ■:ヽ rclこ lncd ‐ r」 cturc but (1) IIIllnerse thc mcmbranc in fresh silver cnhancing solution and agitatc for 10-20 :ゴ 、1‐ ごtural 11un S、sh thoroughly in、 vatcr to stop the reaction.Thc protcin bands v/ill appcar :i nlさ thods as black lines i■ li3 1こ ヽ1ヽ :, lhc:11 1■ 7.3 ■■ouble shooting(membranes) ■■■crcこ nc ヽ ll■ ここく、131: All of the troublc shooting indicators listed abovc in scction 6 apply to membranc (■ tc ==rrf二 incubations.In addition thc following should bc considcrcd for rnembranc incubations

11) Poor labelling

lュ } Chcck transfcr system is working

bl High background is lnasklng spcciic staining

ii l Excessive background

二 Inadcquatc blocklng of transfer membranc(cspCCially nylon based mcmbrancs) Si:uratc for longcr tilnes at higher temperaturcs Usc highcr concentrations of BSA in ll==old cottugatC Includc 0 1%gclatin in thc gold conJugate tO rcduce binding to 、二irlur ヽhsh thc mcmbranc vcry thOroughly beforc thc incubations.

23 (b) SpOntaneous precipitadon of silver due to impure water used for washing membrane.Use only clean distilled water in clean L町 s With no contalmnation. Use a totally negative control silnultancously.

(C) Inadequate washing of membrane fo1lowing the gold incuba」 on.Lengthen the washing procedure in water to rcrnove all salts.

8。 Controls

ltis of greatimportance to perfom controls du五 ng IIIIInunogold labelling procedures in order to conf― thc speciflc labelling in sections and membranes.In addition controls will detcrlmnc the causes ofpoor perfomancc ofindividualreagents or weaknessin any part of the entire procedurc.Some are includcd here:

(a) Omit thC flrst andbody.This will dete― ne if the second gold labellcd andbody is labelling non‐ speciflcally.

(b)Rcplace the pHmary antibody with non― inlmune serum,preferably from the salne anmal before inlmunisation.Ths will deterlmne if ale p壺 mary altibody is speciflc.

(c) Replace the primtt andbody wim an inappropHate antibody fЮ m another species.This will also testthe speciflcity of the pnmary antibody and of the gold conJugate.

(d) Absorb the pnmary antibody with its ttdgen(if avttlable)by addidon of approximately lnM/1 for l hour before the incubatlon.This will also test the

specincity Of the pnm響 .

(C)USe a dttrent goH cottugate d“ Cにd towards a dttrent animal spectt This will detcrlmne if the tissuc has non speciflc attaction for gold particles.

(D USC a knOwn negative tissue.

Where possible a known positive con■ ol should also be included in the serles to demonstrate the validity of the p五 mary antibody and ofthe method.

9. Bibliography

The BBI Catalogue of Research Products carnes a comprehensive list of texts which deal extensively widlinlmunogold labelling.Please request a copy from BBI directly or from your local distribuЮ ■

17.10.97

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