Mouse anti cd105 monoclonal igg

P4 and P40 undifferentiated MSCs were counted and plated in a 96 U-bottom well plate (MidSci) at a density of 1.5 × 10⁵–2.0 × 10⁵ per well. Samples were washed twice with 100 μL in PBS containing 1% bovine serum albumin (BSA; Sigma) and 0.1% sodium azide (Sigma), and centrifuged at 2000 rpm for 1 min. Undifferentiated MSCs were stained with primary antibodies at the following concentrations: rabbit anti-CD90 monoclonal IgG, 1:50; mouse anti-CD105 monoclonal IgG, 1:500; rat anti-CD34 mouse monoclonal IgG; 1:200; and anti-MHC class II monoclonal IgG, 1:500 (Abcam). Cells were incubated on ice for 45 min and centrifuged at 2000 rpm for 1 min. Following incubation, cells were washed one time in PBS/1% BSA/0.1% sodium azide and then re-suspended in secondary antibody (Alexa Fluor^® 488 goat anti-rabbit IgG, Alexa Fluor^® 488 goat anti-mouse IgG, or Alexa Fluor^® 488 goat anti-rat IgG; Life Technologies) at a 1:300 dilution. Samples were incubated on ice for 30 min in the dark. Following secondary staining, samples were washed an additional three times and fixed with 4% paraformaldehyde for 10 min on ice. The cells were re-suspended in PBS/1% BSA/0.1% sodium azide and stored at 4 °C until flow cytometry analysis.

Immunofluorescence staining was performed on the frozen sections of the FVMs by staining with the following antibodies: rabbit anti-EPO polyclonal IgG (1:150 dilution; No. ab126876 Abcam, Cambridge, MA, USA), mouse anti-CD105 monoclonal IgG (1:150 dilution; No. ab69772 Abcam, Cambridge, MA, USA), rabbit anti-VEGF polyclonal IgG (1:200 dilution; No. ab39250 Abcam, Cambridge, MA, USA), tetramethylrhodamine isothiocyanate- conjugated goat anti-mouse IgG (1: 200 dilution; Zhongshan Goldenbridge Biotechnology Co. Ltd., Beijing, China), and/or fluorescein isothiocyanate- conjugated goat anti-rabbit IgG (1: 200 dilution; Zhongshan Goldenbridge Biotechnology Co. Ltd.). The samples were counterstained with 4′,6′-diamino- 2-phenylindole (DAPI) (1: 1,000 dilution, No. D9542; Sigma-Aldrich, St. Louis, MO, USA). All the sections were examined using a fluorescence microscope (DS-Ril-U2; Nikon, Tokyo, Japan) and photographed (DS-U2; Nikon).