Slice Sinking Solution (Store at 4 C, Can Be Used Over an Extended Period of Time) s1

Version 1.00, Jan 15, 2015

Stock Solution

4 % Paraformaldehyde 4 % Paraformaldehyde (from Electron Microscopy Science 16% stock) 1x PBS

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

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

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

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

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

**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.

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).

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)

ExM procedures for brain slices

Perfusion and slicing: Essentially the same as conventional histology.

1. Perfuse with 4% paraformaldehyde. Post-fix the brain in 4% paraformaldehyde (e.g., overnight or as needed).

2. Cut 100 micron brain slices on a vibratome. Then go to the next section, skipping steps 3-5 of this section. OR,

3. Cryoprotect the brain in PBS+30% sucrose+ 100 mM glycine (sinking solution), for about one day until the brain sinks.

4. Freeze the brain using dry ice and 2-methylbutane, and embed brain in OCT, M-1 or other embedding matrix.

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.

Primary antibody staining: Essentially the same as conventional histology.

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.

3. Wash slices with blocking buffer, 4 times, ~30 min each.

ExM specific 2nd antibody staining with DNA labeled antibody.

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).

2. Wash in blocking buffer, 4 times, 30 min each. (2nd antibody wash).

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

4. Wash slices in blocking buffer, 4 times, 10-30mins each.

Gelling:

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.)

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.

Digestion and Expansion:

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.)

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.

Image with conventional fluorescent, confocal microscope, or other desired scopes Chemicals list and suppliers.

Chemical Name Supplier Part Number

ExM Gel or Sodium Acrylate Sigma 408220 Preparation Acrylamide Sigma A9099

N,N′- Sigma M7279 Methylenebisacrylamide

Ammonium Persulfate Sigma A3678

N,N,N′,N′- Sigma T7024 Tetramethylethylenediamine

4-Hydroxy-TEMPO Sigma 176141

Fluorescent Dyes Alexa 488 NHS ester Life Technologies A-20000

Atto 565 NHS Ester Sigma 72464

Atto 647N NHS Ester Sigma 18373

Hybridization Dextran Sulfate Millipore S4030 Buffer SSC Life Tech. 15557

Yeast tRNA Roche 10109495001

Normal Donkey Serum Jackson 017-000-001 Immunoresearch

Fixation and Paraformaldehyde Electron 15710 Permeabilization Microscopy Sciences

Glutaraldehyde Electron 16020 Microscopy Sciences

Triton X-100 Sigma 93426

Glycine Sigma 50046

PBS Life Technologies 70011-044

Protein Digestion Proteinase K New England P8107S Biolabs Ethylenediaminetetraacetic Sigma EDS acid

Guanidine HCl Sigma G3272

Tris-HCl Life Technologies AM9855