Immunohistochemical Analysis of Breast Cancer

IHC was performed on a breast cancer tumor microarray (BR1141A; tissuearray.com). Antibodies used for IHC include pHSF1 (S326; Abcam #76076; RRID: AB_1310328), CD3 (Santa Cruz Biotechnology #sc-20047; RRID: AB_627014), CD8A (CST #85336; RRID: AB_2800052), and CCL5 (Invitrogen #710001; RRID: AB_2532515). IHC was performed by deparaffinization using xylenes followed by rehydration with decreasing alcohol solutions. Slides were then incubated in Bloxall (Vector Laboratories #SP-6000–100) and subjected to antigen retrieval with 10 mmol/L citrate solution in a heated pressure cooker (2,100 Antigen Retriever; Aptum Biologics). Tissues were blocked in animal-free blocker (Vector Laboratories #SP-5035) and incubated in primary antibodies overnight in a humidified chamber. Primary antibodies were detected using Vectastain ABC-HRP kits (Rabbit: PK-4001; Mouse: PK-4010) and developed with DAB Immpact kit (Vector Laboratories #SK-4105) followed by dehydration with increasing alcohol solutions and mounted. Slides were imaged with Motic EasyScan scanner and analyzed with QuPath software (27 (link)).

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Jacobs C., Shah S., Lu W.C., Ray H., Wang J., Hockaden N., Sandusky G., Nephew K.P., Lu X., Cao S, & Carpenter R.L. (2023). HSF1 Inhibits Antitumor Immune Activity in Breast Cancer by Suppressing CCL5 to Block CD8+ T-cell Recruitment. Cancer Research, 84(2), 276-290.

Publication 2023

Bloxall SP-5035 Vectastain ABC-HRP kits

Corresponding Organization :

Other organizations : Indiana University Bloomington, Indiana University Health, Indiana University – Purdue University Indianapolis, University of Notre Dame

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Variable analysis

independent variables

PHSF1 (S326; Abcam #76076; RRID: AB_1310328)
CD3 (Santa Cruz Biotechnology #sc-20047; RRID: AB_627014)
CD8A (CST #85336; RRID: AB_2800052)
CCL5 (Invitrogen #710001; RRID: AB_2532515)

dependent variables

Immunohistochemical (IHC) staining patterns in the breast cancer tumor microarray

control variables

Bloxall (Vector Laboratories #SP-6000–100) for blocking endogenous peroxidase activity
10 mmol/L citrate solution in a heated pressure cooker (2,100 Antigen Retriever; Aptum Biologics) for antigen retrieval
Animal-free blocker (Vector Laboratories #SP-5035) for blocking
Vectastain ABC-HRP kits (Rabbit: PK-4001; Mouse: PK-4010) for primary antibody detection
DAB Immpact kit (Vector Laboratories #SK-4105) for development

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