Ar9 antigen retrievalbuffer

Multiplex immunofluorescence was performed on small cerebellar sections from FFPE
tissue blocks with OPAL reagents from PerkinElmer as described in the
PerkinElmer Multiplex IHC manual. Primary antibodies used are summarized in
eTable 1 (links.lww.com/NXI/A726, method: IF). Pretreatment (heating) in a
Braun household vegetable cooking device was performed in AR9 antigen retrieval
buffer from PerkinElmer for 40 minutes before the first antibody and with AR6
antigen retrieval buffer from PerkinElmer for 30 minutes between each antibody
staining.

Human subject protocol (2007-0511) was approved by Institutional Research Board at M.D. Anderson Cancer Center. Tissue microarrays (TMA) were generated using a Beecher instrument with 0.6 mm cores taken from the donor block and placed into the recipient block in triplicate for each case. Tissue microarrays with triplicate cores for each case were generated from primary RCC. Murine tumor TMA were generated with 5 mm cores. TMAs were immunohistochemically stained and analyzed using inForm software (Caliper Life Sciences). The slides were stained using Opal 4-color IHC Kit (NEL794001KT) from Perkin Elmer. Microwave treatment (MWT) was applied to perform antigen retrieval, quench endogenous peroxidases, and remove antibodies from earlier staining procedures. Perkin Elmer AR6 Antigen retrieval buffer (pH 6) was used for CD8 and PD-1 staining while Perkin Elmer AR9 Antigen Retrieval buffer (pH 9) was used for P-STAT1 staining. The slides were stained with primary antibodies against CD8 and PD-1, corresponding HRP conjugated secondary antibodies, and subsequently TSA dyes to generate Opal signal (CD8, Opal 520; PD-1, Opal 570, or Opal 620). The slides were scanned with the Vectra image scanning system (Caliper Life Sciences), and signals were unmixed and reconstructed into a composite image with Vectra inForm software 2.4.6.