The breast cancer TMA (US Biomax Inc.), including invasive ductal carcinoma cases (grade 2, n = 28; grade 3, n = 25) and medullary carcinoma cases (n = 9), was used for multiplex IHC staining of TRIM21, CD73, CD3, CD8, Ki67, and PanCK by using the Opal multiple color IHC kit (Akoya Biosciences) as described previously (53 (link)). Briefly, the deparaffinized and rehydrolyzed TMA slide was implemented antigen retrieval in AR9 retrieval buffer (Akoya Biosciences), followed by 6 cycles of staining procedures including blocking and binding of primary antibodies and second HRP-linked antibodies and visualized with the corresponding Opal fluorophores. Each staining cycle was finished up with heating in AR6 retrieval buffer (Akoya Biosciences) to release the bounded primary and second antibodies but did not disturb the resident fluorophores. After six-round staining procedures, the slide was counterstained with DAPI. The single-marker staining with individual opal fluorophore was used as the reference for the “spectral unmixing process.” The antibodies and corresponding fluorophores are listed in table S1.
Ar9 retrieval buffer
Manufactured by Akoya Biosciences
The AR9 Retrieval Buffer is a laboratory reagent used to prepare tissue samples for further analysis. It is designed to facilitate the extraction and preservation of target molecules, such as proteins or nucleic acids, from biological samples. The core function of the AR9 Retrieval Buffer is to enable the effective release and recovery of these target analytes from the sample matrix, while maintaining their structural integrity and functionality.
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2 protocols using ar9 retrieval buffer
1
Multiplex IHC Profiling of Breast Cancer
2
Multiplexed IHC Analysis of FFPE Tumor Biopsies
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The formalin-fixed, paraffin-embedded (FFPE) tumor biopsies obtained before (C1D1) and after (C2D8) treatment were analyzed by mIHC staining using the Opal 7-Color Multiplex IHC kit (Akoya Biosciences) as described previously (63 (link)). Briefly, 5 μm FFPE tissue sections were deparaffinized, rehydrated, and refixed with 10% neutral buffered formalin prior to antigen recovery in heated AR9 retrieval buffer (Akoya Biosciences) for 15 minutes. Afterwards, the FFPE sections underwent 6 sequential cycles of staining procedures. Each cycle included blocking, binding of the primary antibody and the corresponding HRP-labeled secondary antibody, and then visualized by a different Opal fluorophore. Each cycle was ended with another heated antigen retrieval process with AR6 retrieval buffer to remove the bound antibody. After the 6-cycle staining procedures, the sections were counterstained with DAPI (Akoya Biosciences) and mounted with Diamond Antifade fluorescence mounting media (Thermo Fisher Scientific). Each single marker with an associated fluorophore staining section served as a reference control in the spectral library for the “spectral unmixing process,” and the unstained slide served as the background control. Each biopsy was used for 2 panels of mIHC staining with the antibodies and corresponding fluorophores listed in Supplemental Table 11 .
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