Rabbit monoclonal anti trimethyl histone h3 lys27 antibody

FFPE tissue was sectioned and deparaffinized and antigen retrieval was performed by microwave oven heating in sodium citrate buffer (10 mM, pH 6.0) at 95 °C for 20 min. To block endogenous peroxidase, slides were dipped in 3% H₂O₂ with methanol for 10 min at room temperature. The slides were subsequently blocked in Dako Antibody Diluent (Dako, Glostrup, Denmark) for 10 min at room temperature. The primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. We used rabbit monoclonal anti-histone H3 K27M antibody (RevMAb Biosciences, San Francisco, CA, USA), mouse monoclonal anti-histone H4 antibody (Cell signaling, Beverly, MA), rabbit monoclonal anti-trimethyl-histone H3 (Lys27) antibody (Merck Millipore, Darmstadt, Germany), and mouse anti-Ki-67 antigen antibody (Dako). Secondary antibodies were goat anti-rabbit IgG (H + L) highly cross-absorbed secondary antibody, Alexa fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA), or goat anti-mouse IgG (H + L) highly cross-absorbed secondary antibody, Alexa fluor 488 (Thermo Fisher Scientific). Slides were stained with DAPI using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA). A BZ-X710 microscope (Keyence, Osaka, Japan) was used for fluorescence microscopy.

Cell lysate was extracted from FFPE tissue samples using a Qproteome FFPE Tissue Kit (Qiagen). 15 μg of total protein from each sample was run on 4–12% bis-tris precast polyacrylamide gel (Thermo Fisher Scientific), transferred to PVDF membrane using a wet system and probed overnight with primary antibody. The following primary antibodies were used: rabbit polyclonal anti-Histone H3 antibody (Abcam, Cambridge, MA), rabbit monoclonal anti-histone H3 K27M (RevMAb Biosciences) and rabbit monoclonal anti-trimethyl-histone H3 (Lys27) antibody (Merck Millipore). The blot was then probed with the rabbit HRP-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK). Membranes were revealed using chemiluminescent detection reagent (GE Healthcare) and enhanced signal was detected in CCD imaging system. Protein extracted from the FFPE tumor sample of H3F3A wild-type glioma (IDH1 mutated) case was used as a negative control for H3 K27M and as a positive control for H3K27me3 [19 (link)].