Immunohistochemical Analysis of mH2A2

Specimens were obtained from Icahn School of Medicine at Mount Sinai and considered non-human subject research. Tissue Microarray slides were provided by the NCI cancer Diagnosis program (CDP). Other investigators may have received slides from the same blocks. IHC was performed as described before^{62 (link)}. In brief, 5 μm sections from formalin-fixed paraffin-embedded specimens were deparaffinized, incubated for antigen retrieval with Vector Citrate-Based Antigen Unmasking Solution (Vector Laboratories) in microwave for 10 min, and then exposed to 0.3% hydrogen peroxide to block endogenous peroxidase activity. After blocking with Vector Normal Horse Serum (2.5%) for 20 min, sections were incubated at 4 °C overnight with mH2A2 (1:350–1:500) prepared in 0.1% BSA. Slides were subsequently developed using Vector imPRESS Universal Kits anti-mouse/rabbit Ig or anti-goat Ig (Vector Laboratories), Vector DAB Peroxidase Substrate Kit as the chromagen (Vector Laboratories) and Harris Hematoxylin (Sigma) for counterstaining. Slides were then sealed and mounted with Permount (Sigma) and randomized for subsequent blinded review.