Mar 14, 2023
Immunohistochemistry of liver tissue sections
Forked from Immunohistochemistry of liver tissue sections
- 1Columbia University
Protocol Citation: Presha Rajbhandari, Taruna Neelakantan, Brent R. Stockwell 2023. Immunohistochemistry of liver tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqjpkxvk5/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: March 14, 2023
Last Modified: March 14, 2023
Protocol Integer ID: 78790
Keywords: IHC, immunohistochemistry
Funders Acknowledgement:
NIH
Grant ID: 4UH3CA256962
Abstract
This protocol outlines the steps used to perform standard immunohistochemistry for antibody validation in frozen human liver tissue samples, performed at Molecular Pathology Core facility at Columbia University.
Cryosection the frozen liver tissue at 5µm thickness and place it on charged slide
Air dry the sections for 3 minutes 03:00:00
Fix the tissue sections in cold acetone for 15 minutes 00:15:00 . Alternatively, fix with 1% paraformaldehyde at 4C for 15min followed by ice-cold methanol at -20C for 5min.
15m
Air dry the sections at room temperature for 2 minutes 00:02:00
Incubate the slides in 0.3% hydrogen peroxide in PBS for 5 minutes 00:05:00 to block peroxidase activity
Wash slides with PBS 3 times, for 5 minutes each 00:05:00 X3
- Block the tissue sections in 10% normal goat serum or 5%Horse serum with 0.1%BSA for 20 minutes 00:20:00
Incubate the tissue sections with primary antibody diluted in DAKO antibody diluent at room temperature for 1.5-2 hours 01:30:00 - 02:00:00
Wash the slides with PBS 3 times, for 5 minutes each 00:05:00 each - 3 times
Incubate the tissue sections with biotinylated secondary antibody diluted in PBS at room temperature for 30-45 minutes 00:30:00 - 00:45:00
Wash the slides with PBS 3 times, for 5 minutes each 00:05:00 each - 3 times
Incubate the tissue sections with ABC (Avidin-Biotin complex) peroxidase solution at room temperature for 30 minutes 00:30:00
Wash the slides with PBS 3 times, 5 minutes each 00:05:00 each - 3 times
Incubate the tissue sections with DAB (3,3’-diaminobenzidine) peroxidase substrate solution until desired color intensity is reached and immerse slides in distilled water
Counterstain with hematoxylin and rinse with distilled water
Dehydrate the sections using 95% ethanol followed by 100% ethanol
Clear with xylene and mount coverslip using mounting medium