Mar 18, 2025
Version 1
TEM processing for cytoskeleton and matix preservation in tissues V.1
- 1University of Manchester
Protocol Citation: Aleksandr Mironov 2025. TEM processing for cytoskeleton and matix preservation in tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8erj8l2w/v1
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: Working
Created: March 18, 2025
Last Modified: March 18, 2025
Protocol Integer ID: 124552
Keywords: transmission electron microscopy, epoxy resins, ultrastructure, cytoskeleton, extracellular matrix
Abstract
Processing schedule to allow good cytoskeleton and extracelluar matrix preservation and visibility in tissues. Ultrathin sections can be examined in TEM without post-staining.
Image Attribution
Aleksandr Mironov
Materials
Stock HEM buffer (0.2M) in water:
HEPES 170mM
EGTA 20mM
MgCl2 4 mM
25% glutaraldehyde stock solution (EM grade)
16% formaldehyde stock solution (EM grade)
0.2M cacodylate buffer stock solution (pH7.2)
Tannic Acid (Low Molecular Weight) powder
4% osmium tetroxide solution in water
3% potassium ferrocyanide solution in 0.2M cacodylate buffer (pH 7.2).
5% uranyl acetate stock solution in water
100% Ethanol (analytical grade)
100% Acetone (analytical grade)
MilliQ grade water
Epoxy resin kit (valid for TAAB LV or TAAB 812)
Safety warnings
Wear protective gear (gloves, labcoat, goggles) when working with toxic materials.
Before start
All tissue pieces have to be cut to small size of about 1-3mm cubed to get enough staining. One of the dimensions should not be more than 1mm, others can be 2-3mm.
Tissue should be dissected as soon as possible after excision from an animal to avoid hypoxia artifacts.
Fixation
Fixation
1h 15m
1h 15m
Fix with 2.5% glutaraldehyde and 4% formaldehyde in 0.1M HEM buffer (pH 7.2) for at least 1
hour.
1h
Rinse with 0.1M HEM buffer (pH 7.2) 3x5min.
15m
Post-fixation
Post-fixation
4h 45m
4h 45m
Incubate with freshly made mixture of 1% osmium tetroxide and
1.5% potassium ferrocyanide in 0.1M cacodylate buffer (on rotator).
1h
Rinse with distilled water 3x5min.
15m
Incubate with 1% tannic acid (low molecular weight) in 0.1M cacodylate buffer (pH7.2) for 1h (on
rotator).
1h
Rinse with distilled water 3x5min.
15m
Incubate in aqueous 1% uranyl acetate solution for at least
2hr (better – overnight).*
2h
Rinse with distilled water 3x5min.
15m
Dehydration
Dehydration
2h
2h
70% ethanol in water (could be left overnight)
20m
90% ethanol in water
20m
100% ethanol
20m
100% ethanol
20m
100% acetone
20m
100% acetone
20m
Epoxy resin infiltration
Epoxy resin infiltration
1d 1h 30m
1d 1h 30m
Prepare fresh resin formulation according to the manufacturer instructions. For better miscibility the resin components (except accelerator) can be warmed up in 60C oven. Mix all components very well by shaking the resin bottle. Leave the bottle with full resin mix in 60C oven for a while (about 10min) to let all bubbles (after shaking) to escape.
30m
Incubate tissue pieces in 25% resin mix in acetone
1h
Incubate tissue pieces in 75% resin mix in acetone
16h
Incubate tissue pieces in 100% resin mix
8h
Polymerization
Polymerization
1d 0h 10m
1d 0h 10m
Put tissue pieces into resin- prefilled silicon moulds.
10m
Log the mould numbers or put sample ID labels (laser printed on small pieces of paper, font 6pt) inside. Top up moulds with resin so the upper surface would be a bit convex.
Put moulds into 60C oven for overnight or longer (can be over a weekend)
1d
Ultrathin sections can be examined in TEM without post-staining.