Mach 4 universal hrp polymer

TMA slides were dual immunohistochemicaly stained by using the NPY (Novus Biologicals, Cat# NB600–1094) and NPY1R (Acris Antibodies, Cat# SP4600P) antibodies. Before the dual staining, NPY antibody was tested in test tissue microarray containing different human prostate tissue samples, NPY1R antibody was tested in LnCap prostate cancer cells transfected with scramble or NPY1R siRNA. Briefly, sections were deparaffinized in xylene, rehydrated through decreasing concentrations of alcohol ending in PBS, subjected to heat-induce antigen retrieval in10 mmol/L citrate buffer (pH 6.0) for 5 minutes, 125 °C in a Pascal instrument (Dako Cat# S280030), and allowed to cool off at room temperature. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide solution in distilled water for 10 minutes at room temperature. To inhibit non-specific staining, sections were incubated with a protein blocking solution (Dako Cat# X0909) for 10 minutes at room temperature, then incubated with rabbit polyclonal antibody against NPY (1:4000, 1 h at room temperature). Sections were washed and the bound antibody was detected by using a Biocare Medical MACH 4 universal HRP-polymer (Cat# M4U534 H) with diaminobenzidine (DAB) as chromogen. To ensure that the second staining will not cross react with the first staining, sections were incubated with a denaturing solution for 3 minutes.