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2.4. Ki‐67 antibody staining and quantification

YY Yitang Yan
JL Jingli Liu
AL Arianna Lawrence
MD Michael J. Dykstra
RF Rick Fannin
KG Kevin Gerrish
CT Charles J. Tucker
ES Erica Scappini
DD Darlene Dixon
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Approximately 3.0 mL of ht‐UtLM and CR‐LM cell cultures (about 75 000 cells) were inoculated into chamber slides (ThermoFisher Scientific, Lab‐Tek Flaskette Chamber Slide system, Cat# 177453) and grown to 80% confluency in Cd‐free culture medium. The slides were fixed in 4% paraformaldehyde in PBS for 10 minutes, rinsed with 1× automation buffer (Biocare Medical Cat# TWB945M), permeabilized in 0.2% Triton X‐100, and washed in 1× automation buffer. Rabbit polyclonal anti‐Ki67 (Biocare Medical Cat#CRM325) antibody was applied to the slides at a dilution of 1:100 for 30 minutes. The slides were then incubated with anti‐Rabbit IgG HRP Polymer (BioCare Medical #MP‐7401) for 30 minutes. The slides were then treated with 3,3‐diaminobenzidine to develop chromogenic reaction. Slides were counterstained with hematoxylin and scanned with Leica Aperio AT2 Digital Whole Slide Scanning system. Three slides for each treatment group were prepared and quantified.

Copyright and License information:  ©2021 Published 2021. This article is a U.S. Government work and is in the public domain in the USA. published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.
This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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