Jan 16, 2024
Version 1
Brain Histology - tissue sectioning and staining V.1
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
Protocol Citation: Yiqin Shen 2024. Brain Histology - tissue sectioning and staining. protocols.io https://protocols.io/view/brain-histology-tissue-sectioning-and-staining-c7ntzmen
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: January 16, 2024
Last Modified: January 16, 2024
Protocol Integer ID: 93619
Abstract
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Sectioning
Sectioning
Turn on and set the water floating device to 37 °C .
Prepare a bucket of ice. Place the samples on ice before sectioning.
Check the blades by doing a few test sections. Change if they are not sharp.
Place a block of sample on the holder.
Adjust the machine so the block is near the blade but not cut. Rotate the handle to bring near the block.
Start sectioning. Avoid ruptures and curling.
Note
If the samples are not cutting well, either check the blade, or place the sample back on ice for a few minutes depending on the situation.
Place the slice on to water with brush, and gently catch them with glass slides. Label each slide well.
Allow the slides to air dry.
Staining- Day 1
Staining- Day 1
Place the slides onto the slide holder, and dip in xylene for 00:30:00 .
30m
Wash the slides with alcohol. The washes are at different concentrations from 100% to 50%, 00:06:00 each.
Rinse with DD H2O
6m
Remove the slides from holder, trace the outside with hydrophobic pen.
Add blocking buffer (Bloxall) to the slides for 00:30:00 .
Rinse with PBS 2X 3min
30m
Place the slides onto the holder. It should be cooked with pressure cooker in citrate buffer for 00:25:00 .
25m
Remove the slides from the holder and place them down.
Add 2.5% horse serum to the slides for 00:25:00 .
Wash with PBS 2X 3 min.
25m
Dilute primary antibody in blocking buffer, add to the slides.
Incubate in the fridge Overnight 4C
25m
Staining- Day 2
Staining- Day 2
2h 21m
Rinse the slides with PBS.
Dip in TBS-T for 00:10:00 for three times.
Rinse with PBS.
10m
Add blocking buffer to the slides for 01:00:00
1h
Add secondary antibody to the slides, at Room temperature 01:00:00 .
1h
Rinse the slides with PBS.
Dip in TBS-T for 00:10:00 for three times.
Rinse with PBS.
10m
Develop color with chromogen, rinse in DD H2O to stop reaction
Put the slides in Hematoxylin for 30s
Rinse 2X DD H2O, and allow the slides to air-dry.
Put the slides in Xylene for 00:01:00 two times.
1m
Coverslip + mounting media