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.