Anti setdb1 antibody

Cells were seeded on slides in a six-well plate. Cells at 80% confluence were washed gently with PBS and fixed at room temperature with 4% polyformaldehyde (MISHU, Xi’an, China) for 15 min. Cells were immersed in 0.5% Triton X-100 prepared in PBS at room temperature for 15 min and were then blocked with normal goat serum in a wet box at room temperature for 30 min. Next, the cells were incubated successively with primary anti-Setdb1 antibody (1:800, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C and with FITC-conjugated secondary antibody (Proteintech, Wuhan, China) in a wet box in the dark at 37 °C for 1 h. Nuclei were stained with DAPI for 5 min, and slides were then stored at 4 °C in the dark. A confocal microscope (Carl Zeiss, Oberkochen, Germany) was utilized to observe cells.

Both bone and other organs were fixed in 4% paraformaldehyde. Tibias and femurs were decalcified in 10% EDTA for 3 weeks. The decalcified specimens were dehydrated in gradient ethanol and embedded in paraffin. After paraffin embedding, sections with a thickness of 5 µm were created along the long axis of the tibia and placed on slides. The sections were placed in an incubator at 37 °C to dry for 72 h. Bone sections were subjected to H&E staining and Goldner’s trichrome staining according to the manufacturer’s protocol. The paraffin sections of the heart, liver, lung, spleen, and kidney were stained with H&E according to the standard protocol. For immunohistochemistry, the sections were incubated overnight with a primary anti-Setdb1 antibody (1:1000, Cell Signaling Technology, Boston, MA, USA) at 4 °C. After incubation with an HRP (horseradish peroxidase)-bound secondary antibody (Beyotime, Shanghai, China), DAB staining and hematoxylin staining (Beyotime, Shanghai, China) were performed. The sections were observed with a light microscope (Nikon, Tokyo, Japan).