Horse serum

In-depth cell phenotyping was performed with multiplex immunostaining as described elsewhere.^{37 (link)} Briefly sequential immunostaining and antibody stripping was performed on 4 µm-thick formalin-fixed paraffin embedded liver tissue sections obtained from all experimental groups (n = 3/group). After deparaffinization with xylene, all tissue sections underwent repetitive cycles of antigen retrieval (Tris-EDTA, pH 9.0, Novus Biologicals, Centennial, CO, USA) for 30 minutes in water bath and antibody stripping using 2-mercaptoethanol/SDS as described previously.^{38 (link)} To avoid nonspecific antibody binding the sections were incubated with 2.5% horse serum (Vector BioLabs, Malvern, PA, USA), for 1 h at room temperature for the first round of staining. Afterward, the sections were incubated overnight at 4°C with primary antibodies (Supplementary table 1S). Whole scanning of sections was performed using Zeiss Axio Observer 7 followed by stitching and background subtraction. The scans were aligned, hyperstacks and concatenated using FIJI HyperStackReg V5.6 plugin. The image segmentation was performed on all binary images using Ilastik software (v 1.3.3). CellProfiler v3.1.9 was used for cell identification, counting and intensity measurement.

Nonspecific antibody binding was prevented by incubating mouse tissues in 2% normal goat serum (Thermo Fisher) and human tissues in 2.5% horse serum (Vector BioLabs, Malvern, PA, USA), for one hour at room temperature. Auto-fluorescence was reduced by incubating the tissue sections in Image-iT FX Signal Enhancer (Thermo Fisher) for 30 min at room temperature.