Version 1.2, March 01, 2015

Stock Solution

4 % Paraformaldehyde

4 % Paraformaldehyde (from Electron Microscopy Science 16% stock)

1x PBS

Slice Sinking solution (Store at 4C, can be used over an extended period of time)

1x PBS

30% (w/v) sucrose

100 mM Glycine

Permeabilization Buffer (Store at 4C, 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 concentration* / Amount (mL) / Final 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).

Materials and Stock Solution Storage:

Once open, we have been storing Sodium Acrylate in an airtight, low humidity or dessicator chamber in -20 degrees C as the solid is moisture sensitive. APS powder and 100% TEMED solution are stored in a room temperature dessicator.

We have been storing the monomer solution mixed up at -20 degrees C for up to 1 month. TEMED, APS, and H-Tempo Stock solutions can be kept in -20 degrees C, and we generally remake the TEMED and APS stocks at least once every 2 weeks.

Slice Gelling Solution: Mix the following 4 solutionson 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 ~200µlof gelling solution. For 200µlgelling solution, mix thefollowing:

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 (TEMEDstock 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

Perfusionand 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 inblocking 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, ~30min each.

ExM specific 2nd antibody staining with DNA labeled antibody.

  1. Incubate slices with DNA-labeled secondary antibodies (10ug/mL) in slice hybridization buffer, for 6-24 hours. (i.e. if using 24 well plate, enough volume cover the brain slice, ~200µlper well).
  2. Wash in blocking buffer, 4 times, 30min each. (2nd antibody wash).
  3. Incubate slices with tri-functional labelin 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 inan Eppendorf tube for 5mins@ 4C, and then replace with new gelling solution for another 25mins. 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., ~200µlof gelling solution for each brain slice. Need ~100µlfor 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 compression of tissue slice (see schematic below). For 30 and 100 µm sections, pieces of #1 coverglasswere 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. 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, and make sure to completely submerge the slice; make sure it does not dry out.) A small slide box, or a plastic well can be used to incubate the gel and digestion buffer.
  2. Washslices 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 plateauafter about the 3rd or 4th wash. The expansion chamber needs to be of adequate size for the sample, we found that for hemislices of mouse brains, the expanded gel fits nicely in a glass bottom 6 well plate (In Vitro Scientific) when the excess gel around the brain is trimmed away (a razor blade works well for this). Gels can be immobilized with 2% low melt agarose in water to prevent drift during imaging.

Image with conventional fluorescent, confocal microscope, or other desired scopes

[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).