Multispectral Imaging for Immune Profiling

Multispectral imaging was performed as described in the supplementary section of Feng et al. (16 (link)) with appropriate optimization. The liver sections were deparaffinized in three changes of xylene and two changes of 100% ethanol and subsequent gradation of 95, 80, and 70% alcohol for 3 min each. After being heat-induced epitope retrieval with a preheated epitope retrieval solution (pH 8.0, Enzo Life Sciences, Inc. USA), endogenous peroxidase was inactivated by incubation in 3% H₂O₂ for 20 min. Next, the sections were pre-incubated with 10% normal goat serum and then incubated overnight with primary antibodies: CD4, Foxp3, T-bet, GATA3, ICOS, and ROR-γt (details in Table S1). The next day, sections were incubated with the HRP-conjugated second antibody (Vector) for 20 min at room temperature. After washing, polymer tagged HRP mediate the covalent binding of a different fluorophore (Opal-520, Opal-570, Opal-620, Opal-650, and Opal-690) sequentially, coupled with tyramide signal amplification (TSA) step as specified by the manufacturer (Perkin Elmer Inc.). At last, sections were counterstained with DAPI (Sigma-Aldrich). Slides were imaged using the PerkinElmer Vectra platform and a 0.3345 mm² area containing at least one portal area was analyzed in batches using PerkinElmer inForm® software and cell quantification of positively stained cells was analyzed by R script.