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Dec 17, 2024

SBF-SEM Sample Preparation Guide

An overview of common SBF-SEM sample preparation recipes

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 CaCl2Post 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 acetate2 h, 50 °C; 1% aqueous uranyl acetate2 x 30 min; ddH2O2 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 resin6 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 ddH2O45 min, RT, 100% ethanol

Resin infiltration:

  • 3 x 45 min, RT, 100% acetoneAt 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 CaCl2Immerse for 2 – 3 h on ice; using same solutionIf 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 prepared0.3 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 thiocarbohydrazide1 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 acetate5 x 3 min, RT; ddH2O30 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 ddH2O10 min, RT; 100% ice-cold acetone

Resin infiltration:

  • 2 h each; 25% Durcupan:Acetone, then 50% D:A, then 75% D:AOvernight; 100% Durcupan2 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 bufferpipette off Osmium, then 2.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer

Washing:

  • 2 x 30 min; distilled water1h, 45°C; 1% aqueous thiocarbohydrazide (TCH)30 min, 45°C; distilled water15 min, RT; distilled water

Second staining:

  • 1.5 h, RT; 2% aqueous OsO4After, pipette off Osmium

Washing:

  • 3 x 10 min, RT; distilled water

En bloc stain:

  • overnight, 4 °C; 1% aqueous uranyl acetate, then 2h, 50°COnce 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 water2 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% QR4-5h 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 sucroseKeep wet during brain removal; same formulaImmersed 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 rocker10 rpm; 0.1 M cacodylate buffer (pH 7.4) with40 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 oxide18 – 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).

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