Rabbit anti opg

After 72 h of dosing and culture, osteoblast climbing tablets were removed and set with 4% paraformaldehyde for 15 min. The samples were subjected to immunofluorescence staining, which was in line with the kit manufacturer’s instructions. The primary antibodies used were mouse anti-RANKL (Proteintech Company, NO 66610-1-Ig, 1:50), rabbit anti-OPG (Abcam Company, NO ab9986, 1:50), rabbit anti-LC3B (Abcam Company, NO #ab51520, 1:100), and mouse anti-Beclin1 (CST Company, NO #4122, 1:100). Diluted anti-rabbit IgG (Santa Cruz Company, NO sc-2004, 1:400) was used as the secondary antibody. After completion of the immunofluorescence reaction, 4′, 6-diamidino-2′-phenylindole (DAPI) was added drop-wise and incubated in the dark for 5 min, and the samples were stained for nuclei. Excess DAPI was washed four times with phosphate-buffered saline (PBS) for about 5 min each. The film was sealed with a solution containing an anti-fluorescence quenching agent, and the collected images were observed under a fluorescence microscope.

Double immunofluorescent staining was performed using the following primary antibodies: mouse anti-NF-κB p65 (dilution ratio: 1:150; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CTSK (1:150; Abcam), rabbit anti-RANKL (1:150; Proteintech Group Inc., Rosemont, IL, USA), rabbit anti-OPG (1:150; Abcam), rabbit anti-OPN (1:200; Abcam), and rabbit anti-OCN (1:200; Abcam). The sections were first blocked using 5% bovine serum albumin and were then incubated with a specific primary antibody, followed by species-matched secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor 594 and Donkey Anti-Rabbit IgG H&L Alexa Fluor 488; Abcam), at a 1:200 dilution. All tissue sections were subjected to autofluorescence quenching (Vector TrueVIEW; Vector Laboratories, Inc.) and mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) following the manufacturer’s protocol. Fluorescent images were acquired using a Leica TCS-SP8 confocal laser scanning microscope (Leica Biosystems, Wetzlar, Germany) within 48 h after mounting. The immunofluorescence expression of each sample was evaluated using the MFI; a.u.).