May 09, 2023
TS Spurrs - Cell Monolayer on Thermanox coverslips (TM - 013)
- 1CSIRO ACDP
Protocol Citation: sandra.crameri 2023. TS Spurrs - Cell Monolayer on Thermanox coverslips (TM - 013). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpk17l5b/v1
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: May 09, 2023
Last Modified: May 09, 2023
Protocol Integer ID: 81635
Keywords: cell monolayer on thermanox coverslip, thermanox coverslip, cell monolayer, resin block for em, resin block, ts spurr, resin
Abstract
Processing a cell monolayer on Thermanox coverslips to Spurrs resin block for EM.
Guidelines
All step times are the minimum time required. Longer steps are acceptable.
Protocol materials
Glutaraldehyde aqueous solution 25% EM Grade ampoulesProSciTechCatalog #C002
Osmium TetroxideProSciTech
Ethanol
ethanol
HEADER
SAN:
SPEC No:
OPERATOR & STEPS:
OPERATOR & STEPS:
Conventional - with reduced times for monlayer thickness
2h 50m
2.5% % volume Glutaraldehyde aqueous solution 25% EM Grade ampoulesProSciTechCatalog #C002 in 0.1 Mass Percent Sorenson's phosphate buffer 07.2 300 mosmol/kg00:20:00 .
20m
Wash buffer 00:05:00
5m
1 Mass / % volume Osmium TetroxideProSciTech in buffer00:30:00
30m
70 % volume Ethanol 00:10:00
10m
95 % volume ethanol 00:05:00
5m
100 % volume ethanol 00:05:00
5m
100 % volume dry ethanol 00:05:00
5m
100 % volume Spurrs:100 % volume dry ethanol in 1:1 ratio00:30:00
30m
100 % volume Spurrs00:30:00
30m
100 % volume Spurrs00:30:00
30m
Oven polymerise 65 °C Overnight