Immunohistochemical Analysis of Breast Tumors

Human breast tumour tissue specimens were obtained following the guidelines approved by the Institutional Review Board at MD Anderson Cancer Center, and written informed consent was obtained from patients in all cases at the time of enrolment. IHC staining was performed as described previously39 (link)42 (link). Briefly, tissue specimens were incubated with antibodies against PD-L1 (Cell Signaling Technology, #13684, 1:30 dilution), p-EGFR (Y1068; #3777, Cell Signaling Technology, 1:200 dilution), p-GSK3β (S9; #9323, Cell Signaling Technology, 1:200 dilution) or Granzyme B (ab4059, Abcam, 1:100 dilution) and a biotin-conjugated secondary antibody and then incubated with an avidin–biotin–peroxidase complex. Visualization was performed using amino-ethylcarbazole chromogen. For statistical analysis, Fisher's exact test and Pearson χ²-test were used and a P value less than 0.05 is considered statistically significant. The intensity of staining was ranked into four groups: high (+++), medium (++), low (+) and negative (−) according to histological scores.