Sep 06, 2022
Light microscopy immunoperoxidase staining protocol
- Yoland Smith1
- 1Emory University:ASAP
Protocol Citation: Yoland Smith 2022. Light microscopy immunoperoxidase staining protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2b6mg5d/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: August 29, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69299
Keywords: Immunoperoxidase, Light microscopy, Staining, ASAPCRN, procedure of light microscopy immunoperoxidase, light microscopy immunoperoxidase, microscopy immunoperoxidase, staining protocol
Abstract
This protocol details the procedure of light microscopy immunoperoxidase staining protocol.
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Light microscopy immunoperoxidase staining protocol
1d 4h 30m
Select sections to process from the brain tissue bank.
Wash sections thoroughly with a phosphate-buffered saline(PBS, 0.01 Mass Percent , 7.4 ) solution.
Treat the sections at Room temperature with a 1% sodium borohydride (NaBH4) solution in PBS for 00:20:00 .
20m
Rinse sections thoroughly in PBS.
Pre-incubate sections for 01:00:00 at Room temperature in a solution containing 1% normal serum (from the species used to generate the secondary antibodies), 0.3% Triton X-100, and 1% bovine serum albumin (BSA) in PBS.
1h
Incubate the sections for 24:00:00 at Room temperature in a solution containing the primary antibodies in 1% normal serum, 0.3% Triton X-100, and 1% BSA in PBS.
1d
Next day, thoroughly rinse the sections in PBS.
Incubate the sections in a PBS solution containing the appropriate biotinylated secondary antibody (1:200; Vector Labs, Burlingame, CA, USA) combined with 1% normal serum, 0.3% Triton X-100, and 1% BSA for 01:30:00 at Room temperature .
1h 30m
Wash the sections in PBS.
Incubate the sections in an avidin-biotin-peroxidase complex (ABC; 1:100; Vector Labs, Burlingame, CA, USA) solution for 01:30:00 at Room temperature .
1h 30m
Rinse the sections in PBS twice followed by a third rinse in TRIS buffer (0.05 Mass Percent ; 7.6 ).
Incubate the sections in a solution containing 0.025% 3,3ʹ-diaminobenzidine tetrahydrochloride, 10 millimolar (mM) imidazole, and 0.005% hydrogen peroxide in Tris buffer for 00:10:00 at Room temperature .
10m
Rinse the sections with PBS.
Mount sections onto gelatin-coated slides, and coverslip with Permount.
Digitalize the slides with an Aperio ScanScope CS system (Aperio Technologies).