Multiplex Immunofluorescence
tumor
TUMOR
Multiplex immunofluorescence (mIF) analysis was conducted on baseline and on-treatment biopsies (obtained at the time of tumor resection). Serial sections from the patient tumor samples were deparaffinized and rehydrated with a series of graded ethanol to deionized water on a BOND RX platform (Leica Biosystems). The full details of the antibodies used, antigen retrieval techniques, antibody dilutions, and incubation times are summarized in Once each target was optimized in the uniplex slides, the Opal multiplexed assay was used to generate multiple staining slides. Primary antibodies were applied to normal human tonsil specimens as controls at optimized concentrations previously determined in uniplex control tissues. Slide Staining was performed consecutively on Leica BOND RX using the same steps as those used in uniplex IF, and the detection for each marker was completed before application of the next antibody. The sequence of antibodies for multiplex staining determined for panel combination was CD8 (opal 480), PDL1 (opal 520), PD1 (opal570), IgG4 (opal 620), CK7 (opal 690), and spectral DAPI (Invitrogen).All fluorescently labeled slides were scanned on Vectra Polaris (Akoya Biosciences) at 40 × magnification using appropriate exposure times, with 8–10 sections selected from each slide for highest resolution imaging (200x). Sections that did not have any antibodies or fluorescent labeling were used to capture the background tissue autofluorescence. Prior to the analysis, all images were assessed for quality control. The criteria for rejection included poor tissue quality (e.g., folded tissue or missing sections) or staining artifacts (e.g., air bubbles, signal dropout, or inadequate washing). The data from the multispectral camera were analyzed using the InForm imaging software (Akoya Biosciences) and Halo (Indica Labs).