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Preprint

ssTEM

NR Nadine Randel
GJ Gáspár Jékely

Last updated date: Apr 14, 2023 Views: 167 Forks: 0

original An abbreviated version of this protocol was published in eLife in May, 2014
Neuronal connectome of a sensory-motor circuit for visual navigation
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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:
  1. Randel, N and Jékely, G(2023). ssTEM. Bio-protocol Preprint. bio-protocol.org/prep2207.
  2. 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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1 Q&A

Thanks for the many detaisl already. What was used as cryoprotectant/cryofiller?
edit 1 Answer 11 Views Apr 14, 2023
This protocol preprint was submitted via the "Request a Protocol" track.