M-IF Staining Protocol
Opal 7-colour kit (NEL811001KT, PerkinElmer) was used for mIF. TMAs were
dewaxed and rehydrated. In the first step, antigen was retrieved at 125
℃ for 3 min and then cooled to room temperature (RT). Washed with TBST
three times for 5min, incubated in H2O2for 10 min. Repeated washed and blocked with blocking buffer. Primary
antibody, PDL-1 (ab237726, abcam, 1:500, dye 480) was incubated at RT
for 30min. Slides were washed and an HRP-conjugated secondary antibody
was incubated at RT for 10min. TSA dye (1:100) was applied for 10min
after washes. The procedures were repeated six times using the following
antibodies, CD3 (ab16669, abcam, 1:200, dye 690; used as T lymphocyte
cell marker [29]), CD8 (ab93278, abcam, 1:100, dye 570; used as
cytotoxic T cell marker [30]), CD56 (ab75813, abcam, 1:500, dye 620;
used as NK cell marker [31]), CD68 (ab213363, 1:1000, abcam, dye
780; used as pan-macrophage marker [32]), programmed death-1 (PD-1)
(ab237728, abcam, 1:300, dye 520), programmed death ligand-1 (PD-L1)
(ab237726, 1:500, dye 480) [33]. Secondary antibodies anti-mouse
(NEF822001EA, PerkinElmer) or anti-rabbit (NEF812001EA, PekinElmer) were
used at a 1:1000 dilution.
Constructing a DiagnosticPrediction Model
By comparing the difference of quantification and spatial distribution
of immune markers, we can construct a diagnostic prediction model by
randomly selected sixty percent of the cases [34,35].