May 09, 2023

TS Procure 812 - primary fixation Karnovsky's - tissue (TM - 013)

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwj342lmk/v1
  • 1CSIRO ACDP
sandra.crameri
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwj342lmk/v1
Protocol Citation: sandra.crameri 2023. TS Procure 812 - primary fixation Karnovsky's - tissue (TM - 013). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj342lmk/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: 81614
Keywords: Procure 812, formalin, epon, procure 812 resin block, tissues to procure, resin block, ts procure, resin, tissue, tm, primary fixation
Abstract
The method is used to process conventionally EM fixed 1mm3 tissues to Procure 812 resin blocks.
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
16% ParaformaldehydeProSciTechCatalog #C004
25% Glutaraldehyde
Osmium TetroxideProSciTech
Ethanol
Propylene oxideProSciTechCatalog #C156
Propylene oxideProSciTechCatalog #C156
Propylene oxideProSciTechCatalog #C156
HEADER
SAN:
SPEC No:
OPERATOR & STEPS:
OPERATOR & STEPS:
Conventional
5h 15m
Karnovsky's fixative: 2.5 % volume 25% Glutaraldehyde and 4 % volume 16% ParaformaldehydeProSciTechCatalog #C004 in 0.1 Mass Percent Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 00:40:00 or overnight

40m
0.1 Mass Percent 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
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