Version 1.4, September 8, 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
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*
38
50
2
29.2
10x
Sodium acrylate
Acrylamide
N,N′-Methylenebisacrylamide
Sodium chloride
PBS
Water
Total
*All concentrations in g/100 mL except PBS
Amount (mL)
2.25
0.5
0.75
4
1
0.9
9.4**
Final
concentration*
8.6
2.5
0.15
11.7
1x
**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:
Sodium acrylate sometimes comes with a variable purity level, which can affect performance.
For every fresh bottle purchased, we make a 38g/100mL (33 wt%) sodium acrylate stock and
check to insure that it is colorless under normal room lighting. If the stock has a yellow tint (see
Figure 1, below), we discard the bottle from which it was made. Once open, we have been
storing sodium acrylate in an airtight, low humidity, or dessicator chamber in -20 degrees C,
since the solid is moisture sensitive. APS powder and 100% TEMED solution are stored in a
room temperature dessicator.
Figure 1. 38g/100mL Sodium Acrylate stock solutions: correct (left) and low purity (right).
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 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):
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)
Add:
Proteinase K (1:100, final concentration 8 units/mL) to digestion buffer before use.
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
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
4. Wash slices in blocking buffer, 4 times, 10-30mins each.
Gel Chamber (side view)
Top Coverglass
Spacer (Coverglass #1/1.5)
Glass Slide
Brain Slice
Figure 2. Gel chamber schematic.
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 (Figure 2) 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
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. 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. 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. 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