Ab76104

4 µm paraffin-embedded tumor serial sections were used to compare Prussian blue staining with CD31, PSMA, and FAP immunohistochemistry. Staining was performed using a rabbit-specific HRP/DAB detection kit (Abcam) following the manufacturer's instructions, with modifications. Briefly, after hydration through a series of ethanol, sections were treated with hydrogen peroxide blocking for 10 min. Antigen retrieval was performed using pH 6 10 mM citrate buffer at 98 °C for 30 min and then incubated with 10% normal goat serum for 1 h for protein blocking. The sections were incubated overnight at 4 °C with anti-CD31 antibody (1:500, Abcam ab182981, EPR17259), anti-FAP antibody (1:300, Abcam ab28244), or anti-PSMA antibody (1:500, Abcam ab76104, EP3253). After washing, sections were treated with biotinylated antibody and streptavidin peroxidase and developed with 3,3′-diaminobenzidine (DAB) (all provided in the kit). Images were processed using a Nanozoomer (Hamamatsu, Japan) and were visualized using ImageJ software (Bethesda, Maryland, USA).
The percentage area of positive staining was calculated by setting color thresholds using ImageJ. Entire images have been adjusted for brightness, contrast, and exposure, consistently between stains.