May 09, 2023
TS Procure 812 - H&E stained section under glass coverslip on glass slide (TM - 013)
- 1CSIRO ACDP
Protocol Citation: sandra.crameri 2023. TS Procure 812 - H&E stained section under glass coverslip on glass slide (TM - 013). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgby3onvpk/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: 81633
Keywords: Procure 812, formalin, epon
Abstract
The method is used to process H&E stained slides through 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
Osmium TetroxideProSciTech
Step 9
Ethanol
In 8 steps
Propylene oxideProSciTechCatalog #C156
Step 14
Propylene oxideProSciTechCatalog #C156
Step 15
Propylene oxideProSciTechCatalog #C156
Step 16
Procure 812 mixProSciTech
In 3 steps
xylene
Step 3
25% Glutaraldehyde
Step 7
HEADER
HEADER
SAN:
SPEC No:
OPERATOR & STEPS:
OPERATOR & STEPS:
Prepare slide
Prepare slide
Using diamond pen etch a circle on the back of the slide to indicate where the BEEM capsule should be placed during embedding.
Remove Coverslip
Remove Coverslip
Remove coverslip by soaking in xyleneContributed by users minimum Overnight , check to see if coverslip has slide off, if not soak another Overnight .
Rehydration
Rehydration
100 % volume Ethanol 00:05:00
5m
95 % volume Ethanol 00:05:00
5m
70 % volume Ethanol 00:05:00
5m
Conventional
Conventional
5h 15m
5h 15m
2.5 % volume 25% Glutaraldehyde in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 00:05:00
5m
0.1 Molarity (M) buffer 00:05:00
5m
1 % volume Osmium TetroxideProSciTech in buffer 00:30:00
30m
70 % volume Ethanol for at least 00:05:00 or overnight
5m
95 % volume Ethanol 00:05:00
5m
100 % volume Ethanol 00:05:00
5m
100 % volume Ethanol dried with molecular sieve 00:05:00
5m
50 % volume Ethanol 50 % volume Propylene oxideProSciTechCatalog #C156 00:10:00
10m
100 % volume Propylene oxideProSciTechCatalog #C156 00:10:00
10m
50 % volume Propylene oxideProSciTechCatalog #C156 50 % volume Procure 812 mixProSciTech 00:20:00
20m
100 % volume Procure 812 mixProSciTech 00:20:00
20m
100 % volume Procure 812 mixProSciTech 01:00:00
1h
Embed & polymerise overnight in oven at 60 °C
To remove the block from the glass slide
To remove the block from the glass slide
9m
9m
Place slide into boiling water on hot plate setting 3 for 00:09:00 .Remove from water, gloves on, use tissues to hold slide down, angle single edged razor blade at 45o underneath tap washer to make a start to removal , then manually break BEEM capsule off slide.
9m