Immunohistochemistry (IHC) was performed as previously described Wang et al., 2017 (link)). Briefly, tumors embedded in paraffin blocks were deparaffinized, and hydrated through an ethanol series. After microwave antigen retrieval in DakoCytomation target retrieval solution pH 6 (Dako), slides were incubated in 0.3% hydrogen peroxide solution in methanol for 15 min at room temperature to inhibit internal peroxidase activity. Next samples were blocked with serum-free protein block solution (Dako) and incubated with corresponding primary antibodies overnight at 4°C. Next day slide s were stained with EnVision+ System–HRP labeled Polymer (Dako) and visualized with DAB peroxidase substrate kit (Vector Laboratories). IHC scoring was performed as described previously (Klein et al., 2001 (link)). Samples with scores more than 4 were considered as “high” expression group.
For frozen sections, fixation of the tissue sections was performed using 4% PFA. Samples were permeabilized with 0.2% Triton-X, blocked with serum-free protein block solution (Dako) and incubated with corresponding primary antibodies overnight at 4°C. Next slides were incubated with Alexa Flour-conjugated secondary antibody for 1 hr at room temperature and mounted in Vectashield mounting medium containing DAPI (Vector Laboratories). Images were captured with Nikon A1 Confocal microscope (Nikon).
For frozen sections, fixation of the tissue sections was performed using 4% PFA. Samples were permeabilized with 0.2% Triton-X, blocked with serum-free protein block solution (Dako) and incubated with corresponding primary antibodies overnight at 4°C. Next slides were incubated with Alexa Flour-conjugated secondary antibody for 1 hr at room temperature and mounted in Vectashield mounting medium containing DAPI (Vector Laboratories). Images were captured with Nikon A1 Confocal microscope (Nikon).