The following mouse anti-human primary antibodies were used for immunocytofluorescence. Mouse anti-human: STRO-1 IgM (Invitrogen), CD73 IgG1 (BioLegend), CD90 IgG1 (BD Pharmingen), CD105 IgG1 (eBioscience, San Diego, CA), CD146 IgG1 (Invitrogen), βIII-tubulin antibodies (Promega corp., Madison, WI), and isotype controls IgG and IgM (both from Invitrogen). Secondary antibodies included goat anti-mouse IgM or IgG1 Alexa Fluor 594 and goat anti-mouse IgG1 Alexa Fluor 488, all from Invitrogen.
Cells grown in chamber glass slides (8 wells) or in culture plates were washed and fixed with 100% ice-cold methanol for 7-10 minutes. After PBS washing, cells were blocked with 5% goat serum in PBS or in blocking buffer (32.5 mM NaCl, 3.3 mM Na2HPO4, 0.76 mM KH2PO4, 1.9 mM NaN3, 0.1% [w/v] bovine serum albumin (BSA), 0.2% (v/v) Triton-X 100, 0.05% (v/v) Tween 20, and 5% goat serum) for 30 min. The primary antibody was then added directly to cells and incubated for 1 hour at room temperature, washed with PBS for 3 times each 5 minutes on a rocker. After PBS wash, secondary antibody (Alexa Fluor 594 or Alexa Fluor 488) in blocking buffer was added and incubated for 1 hour at room temperature in dark. Subsequently, cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 3 minutes. Images were analyzed under a fluorescence microscope.
Cells grown in chamber glass slides (8 wells) or in culture plates were washed and fixed with 100% ice-cold methanol for 7-10 minutes. After PBS washing, cells were blocked with 5% goat serum in PBS or in blocking buffer (32.5 mM NaCl, 3.3 mM Na2HPO4, 0.76 mM KH2PO4, 1.9 mM NaN3, 0.1% [w/v] bovine serum albumin (BSA), 0.2% (v/v) Triton-X 100, 0.05% (v/v) Tween 20, and 5% goat serum) for 30 min. The primary antibody was then added directly to cells and incubated for 1 hour at room temperature, washed with PBS for 3 times each 5 minutes on a rocker. After PBS wash, secondary antibody (Alexa Fluor 594 or Alexa Fluor 488) in blocking buffer was added and incubated for 1 hour at room temperature in dark. Subsequently, cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 3 minutes. Images were analyzed under a fluorescence microscope.