Oct 10, 2024
Version 2
HPF-FS of Solarion arienae for transmission electron microscopy V.2
- 1Charles University, Faculty of Science, Department of Zoology, Prague, Czechia
- SUM-K
Protocol Citation: Marek Valt 2024. HPF-FS of Solarion arienae for transmission electron microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzp15lzp/v2Version created by Marek Valt
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 07, 2024
Last Modified: October 10, 2024
Protocol Integer ID: 109647
Abstract
This is an optimized version of the protocol for standard HPF-FS fixation for transmission electron microscopy that was used for the fixation of the culture of Solarion arienae.
Cell harvest
Cell harvest
Centrifuge a well-grown culture at 1500 g at 4 °C.
5m
Discard supernatant and collect pellet.
Cryo-immobilization by high-pressure freezing
Cryo-immobilization by high-pressure freezing
The obtained pellet was cryo-immobilized by high-pressure freezing (Leica EM ICE) in gold-plated, 3 mm wide copper carriers with a cavity of 0.1 mm (Wohlwend GmbH).
After high-pressure freezing, the samples were transferred to precooled (-90 °C) fixative medium (2% OsO4 in 100% acetone) and processed by automatic freeze substitution (Leica EM AFS), as follows:
incubation at -90 °C for 96 hours
4d
warm up to -20 °C (5°C/hour for 14 hours)
14h
incubation at -20 °C for 24 hours
1d
warm up to 4°C (3°C/hour for 8 hours)
8h
Incubation at 4°C for 18 hours
18h
After AFS, the samples were transferred to room temperature and washed with 100% acetone (3x).
After washing, infiltrate sequentially for 1h each with:
resin-acetone mixture 1:2
1h
resin-acetone mixture 1:1
1h
resin-acetone mixture 2:1
1h
Finally, the samples were infiltrated with pure resin overnight (EMbed 812)
Lastly, polymerize for 48 hours at 60 °C
2d
Section preparation
Section preparation
Cut sections.
For Solarion HPF-FS, 90nm thick sections were cut with a diamond knife on an an EM UC6 (Leica) ultramicrotome.
post-contrast with uranyl acetate and lead citrate.
Lastly, coat with a carbon layer.