An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells
Abstract
1. Introduction
2. Materials and Methods
- Primary Dermal Fibroblast; Human, Neonatal - ATCC, Virginia, USA, Cat. No. PCS-201-010™;
- Essential 8™ Medium—Life Technologies, Waltham, MA, USA 02451, Cat No. A1517001;
- DPBS no calcium, no magnesium—Life Technologies, Waltham, MA, USA 02451, Cat No. 14190144;
- UltraPure™—0.5M EDTA, pH 8.0, Life Technologies, Waltham, MA, USA 02451, Cat No. 15575020.
- Glutaraldehyde—Sigma-Aldrich, Taufkirchen, Germany, Cat. No. G5882;
- Potassium hexacyanoferrate (III)—Warchem, Poland, Cat. No. 52731;
- Osmium tetroxide—Serva Cat. Heidelberg, Germany, No. 31251.03;
- Thiocarbohydrazide—Sigma-Aldrich, Taufkirchen, Germany, Cat. No. 223220;
- A 1% aqueous uranyl—SPI supplies, West Chester, USA Cat. No. 02624-AB;
- Lead nitrate—Supelco Taufkirchen, Germany, Cat. No. 1.07398;
- L- aspartic acid—Sigma-Aldrich, Taufkirchen, Germany, Cat. No. A9256;
- di-Sodium hydrogen phosphate dodecahydrate pure p.a.—Alchem, Poland, Cat. No. 363-117992809;
- Sodium dihydrogen phosphate monohydrate pure p.a.—Alchem, Poland, Cat. No. 363-117991804;
- Epoxy Embedding Medium—Polysciences, Hirschberg an der Bergstrasse, Germany Cat. No. 08792.
3. Procedure
- Fix the cell pellet in 2.5% glutaraldehyde prepared in 0.1 M phosphate buffer (pH 7.4) at room temperature for a minimum of 2 h. (The fixation time can be extended if necessary.)
- Wash the sample in 0.1 M phosphate buffer (pH 7.4) five times for 3 min each.(Note: If fixation time was extended, extend the washing steps accordingly.)
- Incubate the sample in a 1:1 mixture of 3% potassium ferrocyanide (in 0.1 M phosphate buffer) and 4% aqueous osmium tetroxide for 1 h on ice.
- While step 3 is ongoing, prepare the 1% thiocarbohydrazide (TCH) solution: Dissolve 0.1 g TCH in 10 mL of ddH2O. Incubate the solution at 60 °C for 1 h, swirling every 10 min to aid dissolution. Filter the solution through a 0.22 µm Millipore syringe filter. Note: TCH solution must always be freshly prepared before use.
- After step 3, wash the sample in ddH2O five times for 3 min each.
- Incubate the cell pellet in 1% TCH solution for 20 min at room temperature.
- Wash the sample again in ddH2O five times for 3 min each.
- Incubate the sample in 2% aqueous osmium tetroxide for 30 min at room temperature.
- Wash the sample in ddH2O five times for 3 min each.
- Add 1% aqueous uranyl acetate to the sample and incubate overnight at 4 °C (in the dark).
- Wash the sample in ddH2O five times for 3 min each at room temperature.
- While washing, prepare En bloc Walton’s lead aspartate solution. First, make a 0.03 M aspartic acid solution; this acid will dissolve faster as the pH will be lower. En bloc Walton’s lead aspartate solution consists of 0.066 g of lead nitrate in 10 mL of a 0.03 M aspartic acid solution. Adjust pH to 5.5 with 1 N KOH. Incubate the mixture at 60 °C for 30 min. While in incubation, no precipitate should form.
- Return to step 1. After incubation, wash the sample in ddH2O five times for 3 min each at room temperature.
- Add to the cells pellet En bloc Walton’s lead aspartate solution and incubate at 60 °C for 30 min.
- Wash the sample in ddH2O five times for 3 min each.
- Dehydrate the cell pellet in a graded ethanol series for 15 min at each step: 30%, 50%, 70%, 80%, and 96% ethanol, followed by four changes of 100% ethanol (15 min each).
- Incubate the sample for 15 min in a 1:1 solution of acetone and ethanol, then twice for 15 min in 100% acetone.
- After dehydration, transfer the samples to a 50% epoxy embedding medium in acetone and incubate for 2 h. Then, place the sample in an incubator at 56 °C overnight to allow the acetone to evaporate.
4. Results
5. Discussion
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
CLEM | correlative light and electron microscopy |
iPSCs | induced pluripotent stem cells |
SBEM | serial block face electron microscopy |
SEM | scanning electron microscopy |
STEM | scanning transmission electron microscopy |
TEM | transmission electron microscopy |
TCH | thiocarbohydrazide |
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Diak, N.; Chajec, Ł.; Fus-Kujawa, A.; Bajdak-Rusinek, K. An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells. Methods Protoc. 2025, 8, 90. https://doi.org/10.3390/mps8040090
Diak N, Chajec Ł, Fus-Kujawa A, Bajdak-Rusinek K. An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells. Methods and Protocols. 2025; 8(4):90. https://doi.org/10.3390/mps8040090
Chicago/Turabian StyleDiak, Natalia, Łukasz Chajec, Agnieszka Fus-Kujawa, and Karolina Bajdak-Rusinek. 2025. "An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells" Methods and Protocols 8, no. 4: 90. https://doi.org/10.3390/mps8040090
APA StyleDiak, N., Chajec, Ł., Fus-Kujawa, A., & Bajdak-Rusinek, K. (2025). An Optimized Protocol for SBEM-Based Ultrastructural Analysis of Cultured Human Cells. Methods and Protocols, 8(4), 90. https://doi.org/10.3390/mps8040090