Supplementary Figure S2 from T-cell Subset Features and Distributions Evolve across the Colorectal Precancer–Cancer Spectrum

Supplementary Figure S2. Protocol for multiplex immunofluorescence staining for T cells and epithelial cells. Steps 1-4 were manually conducted. Steps 5-9 were performed with a Leica Bond RX Research Stainer (Leica Biosystems, Buffalo, Illinois, U.S.), regarding the condition of primary antibodies and fluorescence dyes (see the Supplementary Table S2). A. Deparaffinization in xylene (Fisher Chemical, Hampton, New Hampshire, U.S.) and rehydration through a graded dehydrated ethyl alcohol series (100% x3 + 95% x1 + 80% x1 + diluted water x5). B. Antigen retrieval with 2100-Retriever (62700-10, Electron Microscopy Science, Hatfield, Pennsylvania, U.S.) in ethylenediaminetetraacetic acid (EDTA) buffer pH 9.0 (S2367, Dako, Copenhagen, Denmark). C. Opal Antibody Diluent/Block (ARD1001EA, Akoya Biosciences, Marlborough, Massachusetts, U.S.). D. Opal polymer HRP Mouse + Rabbit (ARH1001EA, Akoya Biosciences). E. BOND epitope retrieval solution 1&2 (Leica Biosystems). F. Spectral DAPI (FP1490, Akoya Biosciences, Marlborough, Massachusetts, U.S.). G. Cover slips 24x50 mm No.1 thickness (12450S, Epredia, Portsmouth, New Hampshire, U.S.), prolong gold antifade reagent (P36930, Life Technologies Corporation, Eugene, Oregon, U.S.). Research Resource Identifiers (RRIDs) for primary antibodies were listed at Supplementary Table S2, but RRIDs for Opal dyes, staining solutions, and Akoya Biosciences instrumentation had not been assigned.