Vectasheild antifade mounting medium with dapi

FFPE Sections were dewaxed, rehydrated and pre-treated as described above. Sections were then incubated with mouse monoclonal anti-galanin antibody (1:1000, R&D Systems, MAB585) and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:500, Dako, Z0334) diluted in PBS with 2 % goat serum and 0.3 % Triton-X 100 overnight at 4 °C. On the second day, sections were first immersed in PBS (2 × 5 min), then incubated with goat-anti-mouse secondary antibody conjugated with Alexa Fluor® 568 fluorophore (1:200, ThermoFisher Scientific, A-11004) and goat-anti-rabbit secondary antibody conjugated with Alexa Fluor® 488 fluorophore (1:200, ThermoFisher Scientific, A-11008) for 1 h at room temperature. Sections were then rinsed briefly in PBS (3 × 5 min) and incubated in 1 % sudan black B dissolved in 70 % ethanol to block endogenous autofluorescence by lipofuscin, before coverslipping and mounting with VECTASHEILD antifade mounting medium with DAPI (Vector Laboratories, UK).

IF was optimized for proper antigen retrieval and antibody concentrations using placenta, which is positive for both HHLA2 and PD-L1. Slides were baked then underwent subsequent antigen retrieval, de-paraffinization, rehydration, and blocking in normal goat serum block (Santa Cruz) prior to antibody staining, in the same manner as for IHC. Slides were then incubated for one hour in HHLA2 primary antibody followed by a second one-hour incubation in anti-mouse Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific). The slides were subsequently incubated for one hour in PD-L1 or CD45 primary antibody followed by a second one-hour incubation in anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Thermo Fisher Scientific). Slides were coverslipped using VECTASHEILD Antifade Mounting Medium with DAPI (Vector).