Dec 02, 2022
ProExM
This protocol is a draft, published without a DOI.
- 1Bordeaux Imaging Center
- Mónica Fernández Monreal: Modified from Ed Boyden and Paul Tillberg
Protocol Citation: Mónica Fernández Monreal 2022. ProExM. protocols.io https://protocols.io/view/proexm-cjzeup3e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 02, 2022
Last Modified: December 02, 2022
Protocol Integer ID: 73478
Abstract
Classical protocol of protein pre-labeling Expansion- ProExM
(c) Ed Boyden
Materials
| Chemical Supplies | Product | Supplier | Cat Number | |
| ExM Gel or Preparation | Sodium Acrylate* | Sigma | 408220-25g | |
| Acrylamide | Sigma | A4058 | ||
| N,N′-Methylenebisacrylamide | Sigma | M7279 | ||
| Ammonium Persulfate | Sigma | A3678 | ||
| N,N,N′,N′-Tetramethylethylenediamine (TEMED) | Sigma | T7024 | ||
| 4-Hydroxy-TEMPO | Sigma | 176141 | ||
| Acryloyl-X-SE | Life Technologies | A20770 | ||
| NaCl | Sigma | |||
| Fixation and Permeabilization | Paraformaldehyde | Electron Microscopy Sciences | 15714 | |
| Glutaraldehyde | Electron Microscopy Sciences | 16020 | ||
| Triton X-100 | Sigma | 93426 | ||
| Glycine | Sigma | 50046 | ||
| PBS | Life Technologies | 70011-044 | ||
| Protein Digestion | Proteinase K | Sigma Aldrich | P2308-10 mg | |
| Ethylenediaminetetraacetic acid | Sigma | EDS | ||
| Guanidine HCl | Sigma | G3272 | ||
| Tris-base | Life Technologies | |||
| Non Chemical Supplies | Biopsis punch (3-5 mm) | |||
| Brushes |
Gel and disgestion
Monomer solution17 steps
| A | B | C | D | |
| Component | Stock concentration | Final concentration | Amount (mL) | |
| Sodium acrylate | 38 % | 8.6 % | 2.25 | |
| Acrylamide | 40 % | 2.5 % | 0.625 | |
| N,N′-Methylenebisacrylamide | 2 % | 0.15 % | 0.75 | |
| Sodium chloride | 29.2 % | 11.7 % | 4 | |
| PBS | 10x | 1x | 1 | |
| Water | 0.775 | |||
| Total | 9.4 |
Can be aliquoted and kept at -20ºC up to 2-3 weeks.
Fixation
Fixative16 steps
Rat Hippocampal Neurons
4% Paraformaldehyde
4% Sucrose
In PBS
Fluorescence Labeling
Fluorescence Labeling
16h
16h
Fixation: Use fresh 4% PFA solution in PBS (w/ or wo/ 4% sucrose) for 15-20 min. Wash 3 times in PBS and quench aldehydes with glycine or sodium borate.
1h
Permeabilize in blocking buffer (0.2% fish gelatin, 0.1% or 0.3% triton in PBS), 1 hr at RT.
1h
Incubate with primary antibodies in blocking buffer 1h at RT, or overnight at 4ºC.
12h
Wash slices with blocking buffer, 4 times, ~10 min each.
30m
Incubate slice with secondary antibodies in blocking buffer 1 h at RT on a shaker.
1h
Wash with PBS, 3 times, ~10 min each.
30m
Anchoring
Anchoring
6h 30m
6h 30m
Dilute AcX 1:100 (0.1 mg/mL) in 1x PBS (prepare 500 uL/well for 12-well plates).
Wash 2x 15 minutes with PBS before proceeding to gelation. Samples can be stored at 4º C.
30m
Treat stained expressing genetically encoded FPs slices or coverslips for > 6 hours at RT (this reaction can be left overnight).
6h
Gelling
Gelling
2h 40m
2h 40m
Make sure to remove excess PBS from brain slices before incubation with gelling solution. Incubate slices in gelling solution in a 24-well plate for 25 min at 4C. Coverslips with cells can be mounted directly.
25m
Create a humid gel chamber. For 50 um slices, 2 coverglass were used to create a 170 um-depth sandwich chamber. Cell cultures were mounted cells side-down over a drop of gel (35 um for a coverslip of 12 mm). Make sure the slices are flat, and avoid air bubbles trapped inside the chamber.
15m
Incubate 1h or 2h at 37ºC.
2h
Digestion and expansion
Digestion and expansion
13h
13h
Cover the gel in digesting buffer for 15 min and add proteinase K for and overnight incubation @ room temperature (make sure at least 10-fold excess volume of digestion buffer is used, and make sure make it does not dry out).
12h
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: hemislices of mouse brains fit nicely in a 6-well plate when the excess gel around the brain is trimmed away (a razor blade works well for this). 18 mm coverslips can be punched in 4 mm medals before expansion to get gels of around 16 mm.
1h
Mounting the gel
Mounting the gel
35m
35m
Use a cleaned coverslip and add poly-L-lysine 0.1% solution to the top surface for 20-30 min. Rinse with ddH2O and air dry.
30m
Remove excess of liquid around the gel and place the expanded sample on the coverslip. After 20-30 sec, add ddH2O to keep the sample hydrated.
5m