Antibody ab diluent

Twenty-three formalin-fixed paraffin-embedded human HNSCC samples were stained using the Opal 7-Color Automation IHC Kit-50 Slide according to manufacturer’s instructions (Akoya Biosciences) using the following antibodies and antigen retrieval processes: with antibodies against PD-L1 (clone E1L3N, Cell Signaling Technologies cat. 13684S, Opal 620), CD11c (clone 5D11, Sigma Cell Marque cat. 111M-15, Opal 650), CD68 (clone KP1, Dako cat. M0814, Opal 690), and pan-CK (clone AE1/AE3, Dako cat. M351501-2, Opal 540) on a Bond RX autostainer (Leica Biosystems). Slides were dewaxed (Leica), heat treated in ER2 or ER1 antigen retrieval buffer depending on the antibody for 20 min at 93 °C (Leica), blocked in Antibody (Ab) Diluent (Akoya Biosciences), incubated for 30 min with the primary Ab, 10 min with horseradish peroxidase (HRP)-conjugated secondary polymer (anti-rabbit and anti-mouse, Akoya Biosciences), and 10 min with HRP-reactive OPAL fluorescent reagents (Akoya Biosciences). Slides were washed between staining steps with Bond Wash (Leica) and stripped between each round of staining with heat treatment in antigen retrieval buffer. After the final heat treatment in antigen retrieval buffer, the slides were stained with spectral DAPI (Akoya Biosciences), and coverslipped with Prolong Diamond mounting media (Thermo Fisher).

Tissue was fixed in formalin and paraffin-embedded for multiparameter fluorescence immunohistochemistry. Four mm sections mounted on glass slides were sequentially stained for Panel VHuP115: c-fos, TREM1, CA9, CAPS, VIPR2, CD14, Dapi (Akoya). Panel VHuP116: CD64, CD3, HLADR, TREM1, CD206, CD14, Dapi (Akoya) (Table S3). All antibodies were diluted in Biocare antibody dilutant, except the RTU antibodies. Slides were dewaxed (Leica), heat treated in ER2 or ER1 antigen retrieval buffer depending on the antibody for 20 min at 93°C (Leica), blocked in Antibody (Ab) Diluent (Akoya Biosciences), incubated for 30 min with the primary Ab, 10 min with horseradish peroxidase (HRP)-conjugated secondary polymer (anti-rabbit and anti-mouse, Akoya Biosciences), and 10 min with HRP-reactive OPAL fluorescent reagents (Akoya Biosciences). Slides were washed between staining steps with Bond Wash (Leica) and stripped between each round of staining with heat treatment in antigen retrieval buffer. After the final heat treatment in antigen retrieval buffer, the slides were stained with spectral DAPI (Akoya Biosciences), and cover slipped with Prolong Diamond mounting media (Thermo Fisher). Single stain controls, fluorescence-minus-one controls, and appropriate positive and negative control tissues were used throughout the staining process.