2.6. Multiplex immunohistochemistry.
Archival formalin fixed paraffin embedded tissue blocks from CC patients were deposited as sections onto microscope slides. Primary antibodies used were polyclonal rabbit anti-human CD4 (CST), monoclonal mouse anti-human CD25 (Novus), monoclonal mouse anti-human Foxp3 (CST), monoclonal mouse anti-human CD8 (Dako) and polyclonal rabbit anti-human PD1 (CST). Primary CD4, CD25, Foxp3, CD8 and PD1 stains were labeled with EnVisionTM FLEX HRP (Dako) conjugated secondary antibodies followed by tyramide signal amplification (TSATM - plus fluorescein system; Perkin-Elmer) as per manufacturer’s instructions. All immunohistochemistry images were captured using an Evos FL Auto 2 cell imaging system (Thermo Fisher Scientific) and analyses were completed using Image J software . For antigen retrieval, slides were boiled by microwaving in EnVisionTM FLEX target retrieval solution, pH 9.0 (Dako) antigen retrieval buffer for 15 mins. Prior to primary antibody labeling, slides were blocked in a PBS solution containing 2 % goat serum for 30 min. Prior to HRP labeling, slides were blocked using EnVisionTM FLEX peroxidase blocking reagent (Dako). For washing, slides were rinsed with EnVisionTM FLEX wash buffer (Dako) then immersed in wash buffer and agitated for 3 min, then placed into a second wash buffer and agitated for a further 3 min. Each incubation took place in an agitated humid chamber with protection from light where necessary. Following the completion of all antibody labeling, slides were incubated with DAPI for 5 mins, then washed and coverslips were mounted using ProLong GoldTM antifade mountant (Thermo Fisher Scientific) and allowed to set in the dark.