May 12, 2023

Public workspaceTS Procure 812 - primary fixation Karnovsky's - transwells (TM - 013) V.2

DOI
dx.doi.org/10.17504/protocols.io.e6nvwjwpwlmk/v2
  • 1CSIRO ACDP
sandra.crameri
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwjwpwlmk/v2
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 12, 2023
Last Modified: May 12, 2023
Protocol Integer ID: 81816
Keywords: Procure 812, formalin, epon
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
Reagent25% Glutaraldehyde
Step 3
Reagent16% ParaformaldehydeProSciTechCatalog #C004
Step 3
ReagentOsmium TetroxideProSciTech
Step 5
ReagentEthanol
In 6 steps
ReagentPropylene oxideProSciTechCatalog #C156
Step 11
ReagentPropylene oxideProSciTechCatalog #C156
Step 12
ReagentPropylene oxideProSciTechCatalog #C156
Step 13
HEADER
HEADER
SAN:
SPEC No:
OPERATOR & STEPS:
OPERATOR & STEPS:
Conventional
Conventional
5h 15m
5h 15m
All solutions are exchanged above and below the membrane, up to step 10.
Karnovsky's fixative: Concentration2.5 % volume Reagent25% Glutaraldehyde and Concentration4 % volume Reagent16% ParaformaldehydeProSciTechCatalog #C004 in Concentration0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least Duration00:40:00 or overnight

40m
Concentration0.1 Molarity (M) buffer Duration00:15:00

15m
Concentration1 % volume ReagentOsmium TetroxideProSciTech in buffer Duration01:00:00

1h
Concentration70 % volume ReagentEthanol for at least Duration00:20:00 or overnight

20m
Concentration95 % volume ReagentEthanol Duration00:20:00

20m
Concentration100 % volume ReagentEthanol Duration00:20:00

20m
Concentration100 % volume ReagentEthanol dried with molecular sieve Duration00:20:00


20m
Remove membrane
Remove membrane
Remove membrane from transwell scaffold using a 45o scalpel and transfer to a glass vial with Concentration100 % volume
ReagentEthanol dried with molecular sieve.

Conventional
Conventional
5h 15m
5h 15m
Concentration50 % volume ReagentEthanol Concentration50 % volume ReagentPropylene oxideProSciTechCatalog #C156 Duration00:20:00

20m
Concentration100 % volume ReagentPropylene oxideProSciTechCatalog #C156 Duration00:20:00

20m
Concentration50 % volume ReagentPropylene oxideProSciTechCatalog #C156 Concentration50 % volume Procure 812


Concentration100 % volume Procure 812Duration00:20:00

20m
Concentration100 % volume Procure 812 Duration01:00:00

1h
Embed & polymerise overnight in oven at Temperature60 °C , section with diamond knife as usual.