Start preparing your sample for the katana microtome
Osmium Staining for Biological Samples
Osmium based staining is recommended for the best signal to noise ratio in your biological samples1,2. Below is a summary table of the slight overall variations in several of the most popular recent methods.
Yunfeng Hua Protocol - rOTO
Publication: Large volume en-bloc staining for electron microscopy based connectomics3
Recommended for: blocks 0.2-1mm thick
Primary fixation:
15ml 0.15 M cacodylate buffer (pH 7.4)
Fixative mixture: 30ml 0.08 M cacodylate buffer (pH 7.4) + 1.25% glutaraldehyde + 25% paraformaldehyde + 2mM CaCl2
Post fix for 12-24h in fixative mixture
Staining:
1.5 h, RT; 2% aqueous osmium tetroxide with 0.15 M cacodylate buffer to pH7.4
1.5 h, RT; 2.5% potassium ferrocyanide with 0.15 M cacodylate buffer to pH7.4
Washing:
2 x 30 min; ddH2O
Mordant:
45 min, 40°C; 1% unbuffered thiocarbohydrazide
Washing:
2 x 30 min; ddH2O
Second staining:
1.5 h, RT; 2% osmium tetroxide
Washing:
2 x 30 min; ddH2O
En bloc stain:
Overnight, 4 °C; 1% aqueous uranyl acetate
2 h, 50 °C; 1% aqueous uranyl acetate
2 x 30 min; ddH2O
2 h, 50 °C; lead aspartate, pH 5.0
Washing:
5 x 3 min; ddH2O
Dehydration:
30 min each, 50%, 75%,, 100% ethanol
30 min x 3, RT; 100% ice-cold acetone
Resin infiltration:
Overnight, RT; 1:1 acetone:Spurr’s resin
6 h; pure resin with 1% DMAE
Embedding:
48 h, 70 °C; embedding moulds
Song Protocol
Publication: High-contrast en bloc staining of mouse whole-brain and human brain samples for EM-based connectomics5
Recommended for: Human samples, samples up to 2mm thick
Primary fixation:
Following surgical removal, tissue was directly collected in fix solution and kept at 4 °C overnight after slicing into 2mm sections.
Washing:
4 x 30 mins, 0.15 M sodium cacodylate buffer
Post-fixation staining:
22 h, RT; 2% osmium tetroxide in 0.15 M cacodylate buffer (pH 7.4)
Washing:
4 x 30 mins, 0.15 M sodium cacodylate buffer
Iron:
22 h, RT, 2.5% FeCN in 0.15 M sodium cacodylate buffer (pH 7.4)
Second staining:
6 h, RT, 2% OsO4 in 0.15 M CaC (pH 7.4)
Washing:
30 min, RT 0.15 M sodium cacodylate buffer then 3 x 20 min, RT, ddH2O
Mordant:
18h, RT, 2% Pg in water
Washing:
3 x 20 min; ddH2O
Third staining:
6 h, RT, 2% OsO4 in ddH2O
Washing:
3 x 20 min; ddH2O
En bloc stain:
14h, 4 °C; 4% aqueous uranyl acetate, then 2h, 50 °C
Washing:
2 x 20 min, RT; ddH2O
Dehydration:
30 min each, 4 °C; 20%, 40%, 60%, 80% anhydrous ethanol in ddH2O
45 min, RT, 100% ethanol
Resin infiltration:
3 x 45 min, RT, 100% acetone
At 4 °C, Epon resin in acetone; 4h 12.5%, 13h 25%, 4h 37.5%, 4h 50%, 19h 62.5%, 8h 75%, 19h 87.5%, (8h 95%, 19h 95%) x3, (8h 100%, 19h 100%) x2
Embedding:
72h, 60 °C; Epon resin
NCMIR Protocol - OTOTO
Publication: Preparation of Biological Tissues for Serial Block Face Scanning EM (SBEM)4
Recommended for: mammalian tissue <200µm thick
Primary fixation:
2min, 35 °C; perfused with Ringer’s solution + xylocaine (0.2 mg/ml) + heparin (20 units/ml)
5min, 35°C; 0.15 M cacodylate buffer (pH 7.4) + 2.5% glutaraldehyde + 2% paraformaldehyde + 2mM CaCl2
Immerse for 2 – 3 h on ice; using same solution
If required, cut into 100 µm thick sections in ice cold 0.15 M cacodylate buffer with 2 mM CaCl2
Washing:
5 x 3 min; cold cacodylate buffer with 2 mM CaCl2
Post-fixation staining:
1 h, on ice; freshly prepared
0.3 M cacodylate buffer + 3% potassium ferrocyanide with equal amount of 4% aqueous osmium tetroxide
Washing:
5 x 3 min; ddH2O
Mordant:
20 min, RT; 0.22um filtered thiocarbohydrazide
1 h, 35 °C. 0.1 g to 10 mL ddH2O
Washing:
5 x 3 min; ddH2O
Second staining:
30 min, RT; 2% osmium tetroxide in ddH2O
Washing:
5 x 3 min; ddH2O
En bloc stain:
Overnight, 4 °C; 1% aqueous uranyl acetate
5 x 3 min, RT; ddH2O
30 min, 60 °C; lead aspartate; prepared by dissolving 0.66 gm lead nitrate in 10 mL 0.03 M aspartic acid; adjust pH to 5.5, and then oven 30 min 60 °C
Washing:
5 x 3 min; ddH2O
Dehydration:
5 min each, ice cold; 20%, 50%, 70%, 90%, 100%, anhydrous ethanol in ddH2O
10 min, RT; 100% ice-cold acetone
Resin infiltration:
2 h each; 25% Durcupan:Acetone, then 50% D:A, then 75% D:A
Overnight; 100% Durcupan
2 h; fresh 100% Durcupan
Embedding:
48 h, 60 °C; fresh Durcupan
Wickramanayake-Cyzmmek (plant) Protocol
Publication: A conventional fixation volume electron microscopy protocol for plants6
Recommended for: Plant samples
Primary fixation:
2% paraformaldehyde, 2% glutaraldehyde, 0.01% Tween-20, 0.05% malachite green in 0.1M sodium cacodylate buffer (pH 7.4)
2x 15 min vacuum cycles
Overnight, 4°C; 2% paraformaldehyde, 2% glutaraldehyde, 0.1M sodium cacodylate buffer
Washing:
5 x 10 mins; 0.1M sodium cacodylate buffer
Post-fixation staining:
4 h, RT; 1% osmium tetroxide in 0.1 M cacodylate buffer
pipette off Osmium, then 2.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer
Washing:
2 x 30 min; distilled water
1h, 45°C; 1% aqueous thiocarbohydrazide (TCH)
30 min, 45°C; distilled water
15 min, RT; distilled water
Second staining:
1.5 h, RT; 2% aqueous OsO4
After, pipette off Osmium
Washing:
3 x 10 min, RT; distilled water
En bloc stain:
overnight, 4 °C; 1% aqueous uranyl acetate, then 2h, 50°C
Once cool, pipette off uranyl acetate
Washing:
3 x 10 min; distilled water
Lead staining:
2h , 50°C; Walton’s lead aspartate solution at pH 5.5
Washing:
once cool, 3 x 10 min, RT; distilled water
Dehydration:
30 min each, 4 °C; 25%, 50%, 75%, 95%, 100% and 100% anhydrous acetone in distilled water
2 x 30 min; 100% propylene oxide (PO)
Resin infiltration:
2-4h, RT; Quetol 651/NSA embedding resin (QR) in PO; 25%, 33%, 50%, 66%, 75%
2x 4-5h; 100% QR
4-5h then once more overnight, RT; 50ml QR, 1ml DMP-30
Embedding:
48h, 50°C; QR + DMP-30
BROPA Protocol
Publication: High-resolution whole-brain staining for electron microscopic circuit reconstruction7
Recommended for: large volumes ~10mm, e.g. whole mouse brain
Primary fixation:
Perfusion, 30 mL at approximately 0.5 mL/s, freshly prepared 30 min prior; 0.1 M cacodylate buffer (pH 7.2) with 0.25 M (2.5%, w/v) glutaraldehyde and 0.12 M sucrose
Keep wet during brain removal; same formula
Immersed for 48 – 72 h, 2 °C, no agitation
Washing:
5 x 8 – 12 h; 0.1 M cacodylate buffer (pH 7.2) with 0.12 M sucrose
Post-fixation staining:
96 h, RT, dark, gyratory rocker
10 rpm; 0.1 M cacodylate buffer (pH 7.4) with
40 mM osmium tetroxide, 35 mM potassium ferrocyanide and 2.5 M formamide
Mordant:
72 h, RT, dark, gyratory rocker 10 rpm;
0.1 M cacodylate buffer (pH 7.4) with 40 mM osmium tetroxide
Washing:
4 h, RT, dark, gyratory rocker 10 rpm; 0.1 M cacodylate buffer
Second staining:
72 h, RT, dark, gyratory rocker 10 rpm; unbuffered solution of 0.32 M pyrogallol (pH 4.1)
Washing:
4 h, RT, dark, gyratory rocker 10 rpm; 0.1 M cacodylate buffer
En bloc stain:
96 h, RT, dark, gyratory rocker 10 rpm; unbuffered solution of 0.04 M osmium tetroxide
Dehydration:
18 – 24 h each; 10%, 25%, 50%, 75%, 100%, ethanol in water
Resin infiltration:
18 – 24 h; 100% propylene oxide
18 – 24 h each; 25%, 50%, 75%, 100%, modified Spurr’s epoxy in propylene oxide
Embedding:
In custom silicone mold, 48 h, 60 °C;
modified Spurr’s resin formulation
Material Sample Recommendations
Materials of Mohs hardness 3 or less should be suitable for cutting, consult the connectomX team if you are interested in harder materials.
If using a material with empty space or voids, it is recommended it be embedded in resin for optimum cutting, if possible.
It is recommended to use decalcification to prepare tooth or bone samples for SBF cutting8.
References
1. Peddie, C. J. et al. Volume electron microscopy. Nat Rev Methods Primers 2, 51 (2022).
2. Koban, M., Machálková, M. & Javůrek, J. An Integrated Solution for the Complete Serial Block-Face Scanning Electron Microscopy Workflow: From Image Acquisition to Data Processing. Microscopy and Microanalysis 29, 1213–1215 (2023).
3. Hua, Y., Laserstein, P. & Helmstaedter, M. Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6, 7923 (2015).Song, K., Feng, Z. & Helmstaedter, M. High-contrast en bloc staining of mouse whole-brain and human brain samples for EM-based connectomics. Nat Methods 20, 836–840 (2023).
4. J. Deerinck, T., A. Bushong, E., H. Ellisman, M. & Thor, A. Preparation of Biological Tissues for Serial Block Face Scanning Electron Microscopy (SBEM) V2. https://www.protocols.io/view/preparation-of-biological-tissues-for-serial-block-b65drg26 (2022) doi:10.17504/protocols.io.36wgq7je5vk5/v2.
5. Song, K., Feng, Z. & Helmstaedter, M. High-contrast en bloc staining of mouse whole-brain and human brain samples for EM-based connectomics. Nat Methods 20, 836–840 (2023).
6. Wickramanayake, J. S. & Czymmek, K. J. A conventional fixation volume electron microscopy protocol for plants. in Methods in Cell Biology vol. 177 83–99 (Elsevier, 2023).
7. Mikula, S. & Denk, W. High-resolution whole-brain staining for electron microscopic circuit reconstruction. Nature Methods 12, 541–546 (2015).
8. Goggin, P. et al. Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue. Bone 131, 115107 (2020).