Acetylated histone 4 ac h4

Immunofluorescence staining was performed on frozen tissue sections. Prior to immunostaining, the tissue sections were fixed with 4% paraformaldehyde for 15 min and permeabilized for 15 min using 0.5% Triton X-100. The tissue sections were incubated for 1 h at room temperature with blocking solution (5% bovine serum albumin, 10% horse serum, and 0.05% Triton X-100) to block nonspecific binding and then incubated at 4 °C overnight with primary antibodies before being incubated for 1 h with the appropriate secondary antibody conjugated to Alexa Fluor 488 or Cy^TM3 (Jackson ImmunoResearch; West Grove, PA, USA) in the dark. DAPI was used to counterstain the nuclei. The samples were covered with mounting medium (cat. no. P36935; Invitrogen; Thermo Fisher Scientific), overlaid with cover slips, and examined under a confocal laser scanning microscope (Carl Zeiss MicroImaging). The primary antibodies used were as follows: Runx2 (1:100; cat. no. 20700-1-AP; Proteintech; Wuhan, China), histone deacetylase 1 (HDAC1; 1:100; cat. no. 5356; Cell Signaling Technology; Danvers, MA, USA), acetylated histone 3 (AcH3; 1:100; cat. no. 06–599; Millipore; USA), acetylated histone 4 (AcH4; 1:100; cat. no. 06–598; Millipore), and vimentin (1:100; cat. no. 92547; Abcam; Cambridge, MA, USA).