Anti h4k20me3

This was performed as described previously [81 ]. Briefly, formalin-fixed, paraffin-embedded sections were deparaffinized, rehydrated, and blocked for endogenous peroxidases and underwent antigen retrieval according to antibody specifications. Tissues were incubated overnight with the following primary antibodies: anti-human melan A clone A103 (M7196; Dako), anti-H4K20me3 (04–079, Millipore and cs5737, Cell Signaling). Secondary antibodies used for 3,3′-diaminobenzidine (DAB)-based immunohistochemistry were either EnVision + System-HRP Labeled Polymer Anti-mouse (K4001; Dako) or EnVision + System-HRP Labeled Polymer Anti-rabbit (K4003; Dako) based on primary antibody host species. Peroxidase activity was revealed using DAB (K3468; Dako). Samples were then counterstained with hematoxylin, dehydrated, and coverslipped.

The level of trimethylation of histone H3 lysine 4 (H3K4), H3 lysine 9 (H3K9),H3 lysine 27 (H3K27), and H4 lysine 20 (H4K20), dimethylation of H3K9 and H4K20, acetylation of H3K9, H3K27, and H4 lysine 16 (H4K16), and the levels of tuberous sclerosis 2 (TSC2) protein and AKT in the cerebellum of BTBR T+tf/J and C57BL/6J mice was analyzed by Western blot analysis as described in Tryndyak et al.[26] (link). The following primary antibodies, rabbit polyclonal anti-H3K4me3 (Millipore, #07-473), anti-H3K9me3 (Millipore, #07-523), anti-H3K27me3 (Millipore, #07-449), and anti-H4K20me3 (Millipore, #07-463), anti-H3K9me2 (Millipore, #07-212), anti-H4K20me2 (Millipore, #07-367), anti-H3K9ac (Millipore, #06-942), anti-H3K27ac (Millipore, #07-360), anti-H4K16ac (Millipore, #06-762); rabbit monoclonal anti-Tuberin/TSC2 (D93F12) XP (Cell Signaling, #4308)), and anti-protein kinase B (AKT/pan) (C67E7) (Cell Signaling, #4691)) at 1∶1000 dilution were used for immunoblotting.