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
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).
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. 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
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
Chemicals list and suppliers.
ExM Gel or
Preparation
Fluorescent Dyes
Hybridization
Buffer
Fixation and
Permeabilization
Chemical Name
Supplier
Part Number
Sodium Acrylate
Sigma
408220
Acrylamide
Sigma
A9099
N,N′Methylenebisacrylamide
Sigma
M7279
Ammonium Persulfate
Sigma
A3678
N,N,N′,N′Tetramethylethylenediamine
Sigma
T7024
4-Hydroxy-TEMPO
Sigma
176141
Alexa 488 NHS ester
Life Technologies
A-20000
Atto 565 NHS Ester
Sigma
72464
Atto 647N NHS Ester
Sigma
18373
Dextran Sulfate
Millipore
S4030
SSC
Life Tech.
15557
Yeast tRNA
Roche
10109495001
Normal Donkey Serum
Jackson
Immunoresearch
017-000-001
Paraformaldehyde
Electron
Microscopy
Sciences
15710
Glutaraldehyde
Electron
Microscopy
Sciences
16020
Triton X-100
Sigma
93426
Glycine
Sigma
50046
PBS
Life Technologies
70011-044
Protein Digestion
Proteinase K
New England
Biolabs
P8107S
Ethylenediaminetetraacetic
acid
Sigma
EDS
Guanidine HCl
Sigma
G3272
Tris-HCl
Life Technologies
AM9855