The BC tissue microarrays purchased from Shanghai Outdo Biotech Co. Ltd. (Shanghai, China), contained 30 pairs of cancer tumors together with matched adjacent normal tissue. IHC staining was performed for ZBTB7A (Santa Cruz Biotechnology, SC-33683, 1:200) and HIC1 (Proteintech, 24949-1-AP, 1:200) on the same paraffin-embedded tissue blocks used for clinical diagnosis. IHC was performed using the avidin–biotin complex method (Vector Laboratories), including heat-induced antigen-retrieval procedures.
The degrees of immunostaining were reviewed and scored by two independent pathologists. The proportion of the stained cells and the extent of staining were employed as evaluation criteria, with at least 1000 tumor cells evaluated in each case. While one score was assigned based on the percentage of positive cells, another was assigned based on the staining intensity, and the final score was calculated by multiplying the two previous scores.
The degrees of immunostaining were reviewed and scored by two independent pathologists. The proportion of the stained cells and the extent of staining were employed as evaluation criteria, with at least 1000 tumor cells evaluated in each case. While one score was assigned based on the percentage of positive cells, another was assigned based on the staining intensity, and the final score was calculated by multiplying the two previous scores.