Oct 10, 2024

Public workspaceChemical fixation of Solarion arienae for transmission electron microscopy V.2

DOI
dx.doi.org/10.17504/protocols.io.kxygxyd5zl8j/v2
  • 1Charles University, Faculty of Science, Department of Zoology, Prague, Czechia
  • SUM-K
Marek Valt
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DOI: dx.doi.org/10.17504/protocols.io.kxygxyd5zl8j/v2
Protocol Citation: Marek Valt, Pavla Hrubá 2024. Chemical fixation of Solarion arienae for transmission electron microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyd5zl8j/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: September 27, 2024
Last Modified: October 10, 2024
Protocol Integer ID: 109646
Abstract
This is an optimized version of the protocol for standard chemical 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 1250 g at 4 °C.
5m
Centrifigation
Temperature
Discard supernatant and collect pellet.
Chemical cell fixation
Chemical cell fixation
2d 6h 3m
2d 6h 3m
Immerse pellet with 2,5% glutaraldehyde fixative in 0,1M cacodylate buffer solution for 1h. Work on ice.
1h
Toxic
Temperature
Wash with 0,1M cacodylate buffer, 3 times.
Post-fix with 2% OsO4 in 0,1 M cacodylate buffer for 1h, on ice
1h
Toxic
Temperature
Wash fixed samples in distilled water, 3 times.
Dehydrate samples in ethanol series 30%, 50%, 70%, 80%, 90%, 95%, (100% 3 times)
Impregnate the dehydrated sample with acetone 1:1 ethanol solution.
Impregnate with 100% acetone.
Impregnate acetone 1:1 resin (EMbed 812) mixture.
Toxic
Transfer the sample to (EMbed 812) resin. Repeat 3 times every 4h.
(can be left longer or overnight)

4h
Toxic
Polymerize samples embedded in resin at 70 °C for 48h.
2d
Section preparation
Section preparation
Cut sections.
For Solarion, 80nm thick sections were cut with a diamond knife on an Ultracut E ultramicrotome (Reichert).
post-contrast with uranyl acetate and lead citrate.