Immunohistochemical Analysis of Tissue Samples

Four-micrometer sections of formalin-fixed, paraffin-embedded tissues were prepared for conventional IHC and stained for 45 minutes with the rabbit anti-human granzyme B polyclonal Ab (Abcam, ab4059) 1:100, mouse anti-human PD-L1 mAb 10.4 μg/mL (Clone 405.9A11, (Boston, MA)) or rabbit anti-human Ki67 polyclonal Ab 1:2000 (Vector, VP-K451), followed by secondary HRP conjugated anti-rabbit or anti-mouse Ab. The anti-PD-L1 mAb used for IHC [21 (link)] recognizes a different domain than the anti-PD-L1 mAb secreted by the lentivirus. The slides were developed using 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. The quantification of the IHC images was performed using the IHC Profiler Plugin of ImageJ Software [47 (link)].