May 13, 2023
Version 2
TS Procure 812 - primary fixation Karnovsky's - transwells (TM - 013) V.2
- 1CSIRO ACDP
Protocol Citation: sandra.crameri 2023. TS Procure 812 - primary fixation Karnovsky's - transwells (TM - 013). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjwpwlmk/v2Version created by sandra.crameri
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 13, 2023
Last Modified: May 13, 2023
Protocol Integer ID: 81816
Keywords: Procure 812, formalin, epon, procure 812 resin block, transwells to procure, fixed transwell, resin block, ts procure, transwell, resin
Abstract
The method is used to Karnovsky's EM fixed transwells to Procure 812 resin blocks. The presence of 4% paraformaldehyde is only used to satisfy microsecurity requirements.
Guidelines
Sample is not optimal for EM, best to dissect and use only the outer 2mm of tissue mass. Sample is usually derived from histology.
Protocol materials
25% Glutaraldehyde
16% ParaformaldehydeProSciTechCatalog #C004
Osmium TetroxideProSciTech
Ethanol
Propylene oxideProSciTechCatalog #C156
Propylene oxideProSciTechCatalog #C156
Propylene oxideProSciTechCatalog #C156
HEADER
SAN:
SPEC No:
OPERATOR & STEPS:
OPERATOR & STEPS:
Conventional
5h 15m
All solutions are exchanged above and below the membrane, up to step 10.
Karnovsky's fixative: 2.5 % volume 25% Glutaraldehyde and 4 % volume 16% ParaformaldehydeProSciTechCatalog #C004 in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 00:40:00 or overnight
40m
0.1 Molarity (M) buffer 00:15:00
15m
1 % volume Osmium TetroxideProSciTech in buffer 01:00:00
1h
70 % volume Ethanol for at least 00:20:00 or overnight
20m
95 % volume Ethanol 00:20:00
20m
100 % volume Ethanol 00:20:00
20m
100 % volume Ethanol dried with molecular sieve 00:20:00
20m
Remove membrane
Remove membrane from transwell scaffold using a 45o scalpel and transfer to a glass vial with 100 % volume
Ethanol dried with molecular sieve.
Conventional
5h 15m
50 % volume Ethanol 50 % volume Propylene oxideProSciTechCatalog #C156 00:20:00
20m
100 % volume Propylene oxideProSciTechCatalog #C156 00:20:00
20m
50 % volume Propylene oxideProSciTechCatalog #C156 50 % volume Procure 812
100 % volume Procure 81200:20:00
20m
100 % volume Procure 812 01:00:00
1h
Embed & polymerise overnight in oven at 60 °C , section with diamond knife as usual.