Version 1.00, Jan 15, 2015
Stock Solutions
4 % Paraformaldehyde
4 % Paraformaldehyde (from Electron Microscopy Science 16% stock)
1x PBS
Permeabilization Buffer (Store at 4 C, can be used over an extended period of time)
1x PBS
0.1% Triton X-100
Cell Blocking Buffer (Store at 4 C, can be used for at least 1 month)
1x PBS
5% normal donkey serum
Cell 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
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).
Gelation Solution: Mix the following 4 solutions on ice. Monomer solutions + TEMED
accelerator + APS initiator solution + Water (Initiator solution needs to be added last to prevent
premature gelation). Solutions need to be vortexed to ensure fully mixing.
For 200 µL gelling solution, mix the following:
Monomer solution (1.06x) (188µl) (keep at 4C to prevent premature gelation):
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).
Inhibitor solution: not needed for cells, bring up to 200 µL with ddH2O (4 µL).
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 cultured cells
Cell Culture:
Cells can be grown as desired to suit experimental needs. However, for ease of gelation and
imaging, and handling, we have found 16 well Culturewells removable chambered coverglass
from Grace Bio Labs (Sigma catalog: GBL112358-8EA) to be great for before imaging, as well
as subsequent gelation and digestion.
Fixation:
Fixation and permeabilization conditions will depend on immunostaining protocol, we have tried
the procedure with PFA as well as PFA/glutaraldehyde. Below is a standard fixation and
permeabilization protocol.
1.
2.
3.
4.
Fix with cold 4% PFA for 10 min at RT.
Wash with 5 min 1x PBS + 100 mM glycine to quench fixation.
Wash 2x 5 min with 1x PBS
Permeabilize with permeabilization buffer at RT for 15 minutes.
Primary antibody staining: Essentially the same as conventional histology.
1. Block cells with blocking buffer for 15 minutes
2. Incubate cells with primary antibodies in blocking buffer at desired primary
concentration.
a. Can do incubation from 1 hr to overnight at RT/ 4 C.
3. Wash 4x 5 min with PBS.
ExM specific 2nd antibody staining with DNA labeled antibody.
1. Incubate cells with DNA-labeled secondary antibodies (10 ug/mL) in cell hybridization
buffer, for 6 hours.
2. Wash in PBS, 4 times, 5 min each. (2nd antibody wash).
3. Incubate cells with tri-functional label in cell hybridization buffer, for 6 hours. Incubate
DNA trifunctional DNA oligos at 1 ng/uL. 1
4. Wash 4x 5 min with PBS.
Gelation:
Expansion speed is generally limited by the diffusion time of salt and water out/into the gel, thus
casting thin gels will generally speed up expansion time. The Grace Bio labs removable
coverglass has a removable chamber upper structure which can be removed via a removal tool
(Sigma catalog: GBL103259, see Figure 1 below). After removal of the chamber, a 1 mm
silicone spacer remains with the coverslip and can be used to cast the gel (protocol described
below).2 If cells are grown on coverglass, then a chamber similar to the slice gelation chamber
can be made using coverslip spacers (see ExM Slice Protocol).
1. Remove upper structure from the Grace Bio labs chamber leaving silicone gasket on
coverslip.
a. It helps to remove most of the liquid from each well before doing this, leave~ 40
uL of PBS in each well.
2. Prepare a glass slide covered with parafilm to use as the top of the gelation chambers.
a. The parafilm prevents the gels from sticking to the top glass slide.
3. Prepare gelation solution on ice (see above section), adding water and TEMED
accelerator to monomer solution; do not add initiator yet.
a. The volume of each well with 1 mm silicone gasket is ~ 40 uL; prepare 45 uL of
gelation solution for each well.
4. Remove remaining PBS from the wells, make sure to remove all PBS as remaining PBS
will dilute gelation solution.
5. Add APS to gelation solution, vortex briefly, and immediately pipette 40 uL of gelation
solution into each silicone gasket well.
6. Carefully place the parafilm glass slide (parafilm facing down) on top of silicone gasket
to seal the gelation solution within the wells.
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
2
We have found that the top structure can also be removed using a razor blade if one does not have a removal
tool.
a. Done correctly, there should be no bubbles within each well and the gelation
solution will completely fill the volume.
7. Protect sample from dark and move to 37 C incubator for 1 hour for gelation.
Figure 1. Grace bio labs culturewell, with removal tool showing upper structure and bottom
silicone gaskets on coverglass after disassembly (reproduced from Sigma catalog page).
Digestion and Expansion:
1. Digest cells with digesting buffer, overnight @ room temperature. (Make sure at least 10fold excess volume of digestion buffer is used.) With Grace Biolabs chambers:
a. After gelation, carefully remove parafilm slide from top.
b. Then remove black silicone gasket, but keep gels remaining on glass. The black
silicone gasket should peel off easily with tweezers or by hand.
i. Gels will pop off glass surface during digestion on their own; don’t peel
the gels off the surface of the glass.
2. Wash cell gels with excess volume of ddH2O (we usually use at least 10x the final gel
volume), 3-5 times, for 1 hour each time. Gel expansion reaches plateau after about the
3rd or 4th wash.
Then image with conventional fluorescent, confocal microscope, or other desired scopes.
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