Tyramide signal amplification tsa

Sections were dewaxed in xylene (Fisher Chemicals), rehydrated in graded alcohols (Fisher Chemicals), and washed in tap water. Antigen retrieval, hydrogen peroxidase blocking, serum block with NChS, and primary antibody incubation were performed as described for immunohistochemistry. The following day, sections were washed with TBS and incubated with peroxidase labelled secondary antibody (Santa Cruz Biotechnology) at 1:200 in NChS for 30 min, followed by washes in TBS and incubation with Tyramide signal amplification (TSA, Perkin Elmer, Waltham, MA, USA), at 1:50 for 10 min. Antigen retrieval was then performed by microwaving the sections at full power for 2.5 min in 0.01 M citrate buffer, followed by a 30 min cool-down period. The process from NChS block up to primary antibody detection was repeated twice more for two subsequent primary antibodies. Sections were counterstained with DAPI (4′, 6-diamidino-2-phenylindole, Sigma-Aldrich) by adding 1 µL/mL of TBS and incubating the sections for 10 min in the dark. Finally, sections were washed with TBS and mounted with PermaFluor (Life Technologies, Paisley, UK). Primary antibody details are described in Table 1.

Slides of human prostate cancer tissues were obtained and stained under ethics agreement HREC2012.275 (Royal Melbourne Hospital, Melbourne, Australia). Sections were dewaxed, rehydrated and antigen retrieval was performed for 15 min in Citrate buffer (pH 6.10). Endogenous peroxidase activity was blocked using 3% H2O2 in MetOH for 20min at room temperature. Slides were incubated for 2h in blocking buffer alone, followed by an overnight incubation at 4C in blocking buffer containing anti-TROP2 or isotype antibodies (BD Bioscience 551317, 1/500). Biotinylated anti-mouse secondary antibody (1/500, AbCAM) was incubated for 30 min at room temperature, followed by Tyramide Signal Amplification (TSA)(Perkin Elmer, Melbourne, Australia), revelation with diaminobenzidine (DAB) detection kit (AbCAM, Melbourne, Australia), Ethanol/Xylene dehydration and mounting. The percentage of cells considered as positive for TROP2 staining (cf example in Figure 1A) was calculated after counting positive and total cell numbers in 5 randomly chosen fields of approximately 100 tumor cells.