Histone H3K4me3 Immunohistochemistry Protocol

The construction process and detailed information on the HEC tissue microarray (TMA) and immunohistochemistry (IHC) process have been described previously 24 (link),25 (link).Briefly, the slides were deparaffinized and rehydrated in xylene and alcohol gradient, then treated with citric acid epitope retrieval reagent at 100°C for 20 min and cooled to room temperature to prohibit the endogenous peroxidase activity. To block nonspecific binding sites, the sections were incubated with 5% bovine serum albumin (BSA) (YESEN, Shanghai, China) at 37˚C for 30 min. Subsequently, the sections were incubated with primary rabbit anti‑human monoclonal H3K4me3 antibody (#9751, 1:200 dilution, Cell Signaling Technology) overnight at 4°C. The sections were washed with PBS three times and then incubated for 1 hours with horseradish peroxidase (HRP)-labeled secondary antibody (Gene Tech; Shanghai, China). Finally, the sections were stained using diaminobenzidine (DAB) (Gene Tech; Shanghai, China) and imaged by using a microscope (Leica Microsystems Imaging Solutions, Cambridge, UK).