The immunohistochemical staining protocol and semiquantitative analysis were carried out following the protocol of our previous study44 (link). Primary antibodies included anti-Hsp90α (#ab59459, Abcam; Cambridge, MA), anti-ATP1A1 (#ab7671, Abcam; Cambridge, MA) and anti-STAT3 (#ab15523, Abcam; Cambridge, MA).
Following a hematoxylin counterstaining, immunostaining was scored by two independent experienced pathologists, who were blinded to the background information of the mice. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactive score (IRS) was obtained for each case by multiplying the percentage and the intensity score.
Following a hematoxylin counterstaining, immunostaining was scored by two independent experienced pathologists, who were blinded to the background information of the mice. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactive score (IRS) was obtained for each case by multiplying the percentage and the intensity score.
