Anti cd29

Monolayer and single colony-derived adherent cells (at the third passage in culture) were analyzed by immunocytochemistry. Cytospin preparations and growing cells in 6-well culture plate were fixed in 4 % paraformaldehyde for 20 min at 4 °C, washed three times with PBS, and then incubated with 3 % hydrogen-peroxide (H₂O₂; Sigma-Aldrich) at room temperature for 30 min. The cells were permeabilized and pre-incubated with blocking solution (containing 2 % goat serum, 0.3 % Triton X-100, and 0.1 %BSA in PBS) for 30 min at room temperature, and then blocked with 5 % normal goat serum at room temperature for 30 min. Washed slides were separately incubated with anti-CD44 (1:50, Abcam, USA), anti-CD29 (1:50, Abcam, USA) and anti-CD45 (1:50, Abcam, USA) primary antibody at 4 °C overnight. After washing with PBS, Alexa Fluor 488 anti-goat IgG (1:200; Molecular Probes, Carlsbad, CA, USA) was incubated for 1 h at 37 °C. Nuclear staining was performed by treatment with 4′, 6-diamidino-2-phenylindole (DAPI, 1:20,000; Molecular Probes, Carlsbad, CA, USA) for 5 min. Slices were then mounted and observed with a florescent microscope (Leica, Solms, Germany).

Primary antibodies used for immunohistochemical, immunofluorescence, and whole-mount staining included anti-keratin 8 (K8; Progen, Heidelberg, Germany), anti-α-smooth muscle actin (αSMA; Abcam, Cambridge, MA), anti-keratin 77 (K77; Abcam), anti-S100 calcium binding protein A2 (S100A2; Novus Biologicals), anti-CD29 (Abcam), anti-CD49f (Millipore, Milford, MA), anti-platelet endothelial cell adhesion molecule (CD31; Abcam), and anti-protein gene product 9.5 (PGP9.5; Abcam). Secondary antibodies were species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) and species-specific fluorescent dye-conjugated secondary antibodies (Invitrogen, Carlsbad, CA). F-actin was stained with Alexa Fluor 488 and 594 Phalloidin (Invitrogen).

Cells were harvested using 0.05% trypsin-EDTA, centrifuged at 200× g for 5 min, and incubated at room temperature with 5 µL of anti-CD29 (Abcam), -CD90 (eBioscience, Altrincham, UK), -CD105 (AbD Serotec, Altrincham, UK) or -SSEA4 (eBioscience, Altrincham, UK) fluorescently-conjugated antibodies for 30 min. Cells were washed and stored on ice in the dark until analysis using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, High Wycombe, UK). Each surface marker was analysed in triplicates, and the experiment was repeated three times with 50,000 events recorded for each measurement.

BMSCs were isolated from bilateral femurs and tibia of rats, as described previously (Yang et al. 2022 (link)). The BMSCs were cultured in MEM-α (Gibco, 12,571–063, USA) supplemented with 10% FBS (Gibco, 12,664–025, USA), 1% penicillin and streptomycin (Gibco, 15,140–122, USA), and maintained at 37 °C with 5% CO₂. For cell surface marker characterization using flow-cytometry, 1 × 10⁶ cells at passage 3 were stained with 1:100 dilution of fluorescein isothiocyanate3-conjugated anti-CD29 (Abcam, ab27947, UK), CD45 (Abcam, ab10558, UK), and CD90 (Abcam, ab226, UK) antibodies, respectively. Corresponding isotype-matched control antibodies were used for negative controls. Samples were analyzed using the BD FACSCanto™ II Flow Cytometer (BD Biosciences).

Cells grown on coverslips were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin (BSA) and 10% Normal Donkey Serum(NDS) or 10% Normal Goat Serum(NGS) for 1 h at RT^{4 (link)}. The following primary antibodies were then used to incubate with the cells overnight at 4 °C, the neuronal markers: anti-NFM, anti-MAP2, anti-TuJ-1 (1:200, Abcam), anti-NSE and anti-GFAP(1:200, Neuromics, MN, USA); cell surface markers: anti-CD29, CD44 (1:100, Abcam) and CD71, CD73 (1:100, Santa Cruz, CA, USA); the epigenetic markers: anti-histone acH3K9(1:200, Santa Cruz), anti-histone meH3K9(1:200, Abcam), anti-phosphor-Histone H3S10(1:1,000, Santa Cruz); the pluripotent markers: anti-Oct4, anti-Sox2 and anti-Nanog (1:200, Cell Signaling Technology, Danvers, MA, USA)^{4 (link), 18 (link)}. After that, the cells were incubated with CY3/488/543-labeled secondary antibody (Invitrogen, CA, USA) for 1 h, and the results of immunofluorescence were observed under confocal microscopy. The catalog numbers for all primary antibodies used in this research are presented in Table S1.

Cells harvested from SD rats were collected, centrifuged, counted, and adjusted to 1 × 10⁶ cells/ml/tube. Cells were washed by PBS twice and resuspended in 100 μl PBS, then incubated with anti-CD34 (Cat. no. sc-7324; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-CD45 (Cat. no. 202207; Biolegend, San Diego, CA, USA), anti-CD90 (Cat. no. 202526; Biolegend) and anti-CD29 (Cat. no. ab36219; Abcam, Cambridge, UK) antibodies at room temperature in dark for half an hour. A FACS flow cytometer (CytoFLEX; Beckman Coulter, Inc., Brea, CA, USA) was used.