Protocol detail

Immunohistochemistry Staining Protocol

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Immunohistochemistry (IHC) was performed as previously reported^{42 (link)}. Formalin fixed paraffin embedded (FFPE) tissue sections (4 μm) were prepared on Superfrost Plus microscope slides (Thermo Fisher Scientific). Sections were deparaffinized followed by antigen retrieval in CC1 buffer (pH 9, 95°C; Roche), endogenous peroxidase blocking, and then incubation with primary antibodies. Chromogenic detection was performed with VECTASTAIN Elite ABC-HRP kit (Vector Laboratories) followed by counterstaining with hematoxylin. Images were recorded with the Aperio Leica ScanScope XT (Leica Biosystems, Richmond, IL) using x20 objective lens. Primary antibodies used for this study are listed in Supplementary Table S1.

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Borowsky A., Glencer A., Ramalingam K., Schindler N., Mori H., Ghule P., Lee K., Nachmanson D., Officer A., Harismendy O., Stein J., Stein G., Evans M., Weaver D., Yau C., Hirst G., Campbell M, & Esserman L. (2024). Tumor microenvironmental determinants of high-risk DCIS progression. Research Square.

Publication Preprint 2024

Superfrost Plus CC1 buffer VECTASTAIN Elite ABC-HRP kit

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Variable analysis

independent variables

Tissue sections (4 μm)

dependent variables

Immunohistochemistry (IHC) staining patterns
Expression of proteins detected by primary antibodies

control variables

Formalin fixed paraffin embedded (FFPE) tissue sections
Superfrost Plus microscope slides (Thermo Fisher Scientific)
Deparaffinization procedure
Antigen retrieval in CC1 buffer (pH 9, 95°C; Roche)
Endogenous peroxidase blocking
Chromogenic detection with VECTASTAIN Elite ABC-HRP kit (Vector Laboratories)
Counterstaining with hematoxylin
Imaging with Aperio Leica ScanScope XT (Leica Biosystems, Richmond, IL) using x20 objective lens

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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