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ssTEM
Last updated date: Apr 14, 2023 Views: 175 Forks: 0
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This is an updated protocol that works well for Platynereis larvae.
- place the planchettes on a Hexadecane-soaked filter paper before making the planchette sandwich for HPF
- freeze sub medium 1% OsO4 + 0.1% UA in aceton
- Step 1 T-start -90°C, T-end -90°C, time >18h
- Step 2 T-start -90°C, T-end -30°C, slope 5°C/h time 12h
- Step 3 T-start -30°C, T-end -30°C, time 5h
- Step 4 T-start -30°C, T-end 0°C, slope 5°C/h time 6h
- AFS 0°C 4x30 min aceton water free
- AFS 0°C 1x3h Epon 812 - 25% w/o BDMA
- AFS 0°C o/n Epon 812 - 50% w/o BDMA
- AFS room temp 4h Epon 812 - 50% w/o BDMA
- AFS room temp 4h Epon 812 - 75% w/o BDMA
- AFS room temp o/n Epon 812 - 75% w/o BDMA
- AFS room temp 4h Epon 812 - 100% w/o BDMA
- AFS room temp 4h Epon 812 - 100% w/o BDMA
- AFS room temp o/n Epon 812 - 100% w/o BDMA
- AFS room temp 4h Epon 812 - 100% with BDMA
- AFS room temp 4h Epon 812 - 100% with BDMA
- AFS room temp o/n Epon 812 - 100% with BDMA
- polymerization at 66°C minimum 2 days
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
- Randel, N and Jékely, G(2023). ssTEM. Bio-protocol Preprint. bio-protocol.org/prep2207.
- Randel, N., Asadulina, A., Bezares-Calderón, L. A., Verasztó, C., Williams, E. A., Conzelmann, M., Shahidi, R. and Jékely, G.(2014). Neuronal connectome of a sensory-motor circuit for visual navigation. eLife. DOI: 10.7554/eLife.02730
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