<p>Version 1.00, Jan 15, 2015</p><p>Stock Solution</p><p>4 % Paraformaldehyde 4 % Paraformaldehyde (from Electron Microscopy Science 16% stock) 1x PBS</p><p>Slice Sinking solution (Store at 4 C, can be used over an extended period of time) 1x PBS 30% (w/v) sucrose 100 mM Glycine </p><p>Permeabilization Buffer (Store at 4 C, can be used over an extended period of time) 1x PBS 100 mM Glycine 0.1% Triton X-100 </p><p>Slice Blocking Buffer (Store at 4 C, can be used for at least 1 month) 1x PBS 0.1% Triton X-100 5% normal donkey serum</p><p>Slice Hybridization Buffer (Store at 4C, can be used for at least 1 month): 2x SSC buffer (saline-sodium citrate) 10% (w/w) dextran sulfate 1 mg/ml yeast tRNA 5% normal donkey serum 0.1% Triton X-100 </p><p>Monomer solution: Component Stock Amount (mL) Final concentration* concentration* Sodium acrylate 38 2.25 8.6 Acrylamide 50 0.5 2.5 N,N′-Methylenebisacrylamide 2 0.75 0.15 Sodium chloride 29.2 4 11.7 PBS 10x 1 1x Water 0.9 Total 9.4** *All concentrations in g/100 mL except PBS</p><p>**9.4/10 mL (1.06x), the remaining 6% volume brought up by initiator, accelerator and inhibitor as needed (see below). Slice Gelling Solution: Mix the following 4 solutions on ice. Monomer solutions + TEMED accelerator + APS initiator solution + 4-hydroxy-TEMPO (abbreviated 4HT) inhibitor solution. The initiator solution needs to be added last, to prevent premature gelation. Solutions need to be vortexed to ensure full mixing.</p><p>Each slice needs ~200µl of gelling solution. For 200µl gelling solution, mix the following: Monomer solution ( 1.06x ) (188µl) (keep at 4C to prevent premature gelation): Inhibitor solution (4µl): 4-hydroxy-TEMPO (4HT stock solution at 0.5%, final concentration 0.01%) (Inhibits gelation to enable diffusion into brain slices.) Accelerator solution (4µl): TEMED (TEMED stock solution at 10%, final concentration 0.2% (w/w). (Accelerates radical generation by APS). Initiator solution (4µl): APS (APS stock at 10%, final concentration 0.2% (w/w)). (This initiates the gelling process. This needs to be added last). </p><p>Digestion Buffer (can be stored as aliquots in freezer at -20C): Proteinase K (1:100, final concentration 8 units/mL) 50 mM Tris pH 8.0 1 mM EDTA, 0.5% Triton X-100, 0.8 M guanidine HCl (8M guanidine HCl stock solution can be kept at RT)</p><p>ExM procedures for brain slices</p><p>Perfusion and slicing: Essentially the same as conventional histology. </p><p>1. Perfuse with 4% paraformaldehyde. Post-fix the brain in 4% paraformaldehyde (e.g., overnight or as needed).</p><p>2. Cut 100 micron brain slices on a vibratome. Then go to the next section, skipping steps 3-5 of this section. OR,</p><p>3. Cryoprotect the brain in PBS+30% sucrose+ 100 mM glycine (sinking solution), for about one day until the brain sinks. </p><p>4. Freeze the brain using dry ice and 2-methylbutane, and embed brain in OCT, M-1 or other embedding matrix. </p><p>5. Cut brain slices with cryotome. 30 µm slices are typical for antibody penetration, although slices up to at least 100 µm thick are compatible. Store slices in PBS @ 4 C. </p><p>Primary antibody staining: Essentially the same as conventional histology.</p><p>1. Permeabilize slices in blocking buffer, 6 hours, RT. 2. Incubate slice with primary antibodies in blocking buffer, overnight, at RT or 4C on a shaker.</p><p>3. Wash slices with blocking buffer, 4 times, ~30 min each.</p><p>ExM specific 2nd antibody staining with DNA labeled antibody. </p><p>1. Incubate slices with DNA-labeled secondary antibodies (10 ug/mL) in slice hybridization buffer, for 6-24 hours. (i.e. if using 24 well plate, enough volume cover the brain slice, ~200µl per well). </p><p>2. Wash in blocking buffer, 4 times, 30 min each. (2nd antibody wash).</p><p>3. Incubate slices with tri-functional label in slice hybridization buffer, for 6-12 hours. Make sure to cover the slices from light to avoid fluorophore bleaching from this point on. Incubate trifunctional DNA oligos at 1 ng/uL. 1</p><p>4. Wash slices in blocking buffer, 4 times, 10-30mins each.</p><p>Gelling:</p><p>1. Make sure to remove excess PBS from brain slices before incubation with gelling solution. Incubate slices in gelling solution in an Eppendorf tube for 5 mins @ 4C, and then replace with new gelling solution for another 25 mins. Use freshly prepared gelling solution, immediately after adding APS at 4C. (Make sure at least 100-fold excess volume of monomer solution is used. E.g., ~200µl of gelling solution for each brain slice. Need ~100µl for each of two incubations.) </p><p>2. Transfer slices from Eppendorf tube into a gel chamber and then incubate at 37C for 2 hours. Gel chambers are constructed by sandwiching the slice between a slide and a coverglass, with spacers on either side of the tissue section to prevent contact. For 30 and 100 µm sections, pieces of #1 coverglass were used for spacers and for 200 µm sections, pieces of # 1 coverglasses can be stacked two coverglasses thick. (Spacers are easy to make from full coverglasses with a diamond scribe.) Make sure the slices are flat, and avoid air bubbles trapped inside the chamber. </p><p>Digestion and Expansion: </p><p>1 Each DNA-labeled 2nd antibody is conjugated to a 42bp long DNA sequence that contains two 20bp long complementary sequences for two tri-functional labels. We usually prepare both trifunctional oligos premixed at 50 ng/uL (50x stock). 1. Take off the cover of the gel chamber, and submerge it in digesting buffer, overnight @ room temperature. (Make sure at least 10-fold excess volume of digestion buffer is used.)</p><p>2. Wash slices with excess volume of ddH2O (we usually use at least 10x the final gel volume), 3-5 times, for 15mins each time. Slice expansion reaches plateau after about the 3rd or 4th wash. </p><p>Image with conventional fluorescent, confocal microscope, or other desired scopes Chemicals list and suppliers.</p><p>Chemical Name Supplier Part Number</p><p>ExM Gel or Sodium Acrylate Sigma 408220 Preparation Acrylamide Sigma A9099</p><p>N,N′- Sigma M7279 Methylenebisacrylamide</p><p>Ammonium Persulfate Sigma A3678</p><p>N,N,N′,N′- Sigma T7024 Tetramethylethylenediamine</p><p>4-Hydroxy-TEMPO Sigma 176141</p><p>Fluorescent Dyes Alexa 488 NHS ester Life Technologies A-20000</p><p>Atto 565 NHS Ester Sigma 72464</p><p>Atto 647N NHS Ester Sigma 18373</p><p>Hybridization Dextran Sulfate Millipore S4030 Buffer SSC Life Tech. 15557</p><p>Yeast tRNA Roche 10109495001</p><p>Normal Donkey Serum Jackson 017-000-001 Immunoresearch</p><p>Fixation and Paraformaldehyde Electron 15710 Permeabilization Microscopy Sciences</p><p>Glutaraldehyde Electron 16020 Microscopy Sciences</p><p>Triton X-100 Sigma 93426</p><p>Glycine Sigma 50046</p><p>PBS Life Technologies 70011-044</p><p>Protein Digestion Proteinase K New England P8107S Biolabs Ethylenediaminetetraacetic Sigma EDS acid</p><p>Guanidine HCl Sigma G3272</p><p>Tris-HCl Life Technologies AM9855</p>