Fitc conjugated goat anti rabbit igg h l secondary antibody

Exosome isolation reagent (from cell culture media) was purchased from Thermo Fisher scientific (4478359, China). PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling (MINI67-1KT, China) and PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (PKH26GL-1KT, China) were purchased from Sigma. Lipopolysaccharide (LPS, Escherichia coli 0111: B4, L4391), β‐glycerophosphate (G9422), L‐ascorbic acid 2‐phosphate (49752), and dexamethasone (D1756) were from Sigma, China. Antibodies used in this study were rabbit polyclonal antibody against alkaline phosphatase (ALP) (ab108337, Abcam), CD63 Antibody (sc-5275, SANTA CRUZ Biotechnology), CD81 Antibody (sc-166029, SANTA CRUZ Biotechnology), calnexin (2679 T, cell Signaling Technology), Grp94 (20292 T, cell Signaling Technology), and Lamin A/C (4777 T, cell Signaling Technology). Secondary antibody used for immunofluorescence staining was fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H + L) Secondary Antibody (31635, Thermo Fisher Scientific, China).

Immunofluorescence assay was performed to assess osteoblastic activity. According to manufacturer’s instruction, sections were incubated with the primary antibody against OC (sc-30044, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 overnight at 4 °C, followed by incubation with the FITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody (65-6111, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS for 3 h at room temperature. Finally, DAPI was utilized to label nuclei.