Jun 06, 2023
Immunohistochemistry Protocol
- Hong-Yuan Chu1,2
- 1Van Andel Institute;
- 2ASAP
Protocol Citation: Hong-Yuan Chu 2023. Immunohistochemistry Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr417ogmk/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 31, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82682
Keywords: immunohistochemistry, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-020572
Abstract
This protocol describes immunohistochemistry.
Attachments
729-1840.docx
19KB
Materials
Materials
| A | B | C | |
| Reagent | Supplier | Cat. # | |
| Phosphate Buffered Saline, pH 7.4 | Millipore-Sigma | P3813-10PAK | |
| Triton X-100 | Millipore-Sigma | T9284-100ML | |
| Normal Donkey Serum | Jackson ImmunoResearch | 017-000-121 | |
| ProLong Diamond Antifade Mountant | Fisher Scientific | P36970 |
Phosphate buffered saline powder, pH 7.4, for preparing 1 L solutions Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3813
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T9284
Normal Donkey SerumJackson ImmunoResearch Laboratories, Inc.Catalog #017-000-121
ProLong Diamond Antifade MountantThermo Fisher ScientificCatalog #P36970
Preparation
Preparation
Make up 1 L of fresh Phosphate Buffered Saline (PBS) from 10x stock solution.
Make up PBS-T: 0.2–0.5% Triton X-100 in PBS (stir 100-250 mg Triton in 50 mL of PBS).
Primary reactions
Primary reactions
Place each series in a glass vial and rinse mouse brain sections with PBS 3 times before starting primary reactions.
Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL ) to each series and swirl briefly.
Optional blocking step: leave slices in PBS-T and 2% Normal Donkey Serum at Room temperature for 45–60 min.
Add primary antibody to each series.
Shake gently for 48:00:00 at 4 °C (sections should barely revolve around the vial).
2d
Secondary reactions
Secondary reactions
1d 1h 30m
1d 1h 30m
Rinse sections with PBS 3 times before starting secondary reactions.
Add 1 mL of PBS-T with 2% Normal Donkey Serum (20 µL ) to each series and swirl briefly.
Add secondary antibody to each series.
Shake gently for 01:30:00 at Room temperature , protected from light (sections should barely revolve around the vial).
1h 30m
Rinse sections with PBS 3 times before mounting.
Mount sections serially on slides with Prolong Diamond Anti-fade mounting media; protect slides from light and keep at 4 °C after 24:00:00 drying at Room temperature .
1d