Supplementary Fig. 2: Multi-antibody qualification of PD-L1 and FOXP3 antibodies by t-CyCIF in human FFPE tonsil tissue.

Representative images of immunofluorescence staining from human FFPE tonsil sections using a) three different antibodies for PD-L1: E1L3N, CST; 22C3, DAKO; and 28-8, Abcam (Scale bars: 100 µm). b) Plots of the pixel-by-pixel correlation of the signal intensity generated by the various PD-L1 antibodies. c) Immunofluorescence staining with two different antibodies for FOXP3: 23A/E7, eBioscience; and 206D, BioLegend (Scale bars: 100 µm). d) Plots of the pixel-by-pixel correlation of the signal intensity generated by the various FOXP3 antibodies. The plots in b and d display the correlation of a random sampling of 2,000 pixels. The plots on the lower left (with blue dots) show the original fluorescence intensity at each pixel, and the plots on the upper right (with cyan dots) show the log-transformed fluorescence intensity at each pixel. The Pearson correlation coefficients (R) are shown. DR, dynamic range. The dynamic range and correlation values from 5 different regions are presented in Supplementary Table 1.