Cd105 pe

For flow cytometry analysis, oBMSC were stained with the following anti-human antibodies: CD34-APC, CD90-FITC, CD73-APC, CD105-PE, CD146-APC, CD271-PE, HLA-DR-PE, and isotype controls (Miltenyi Biotec). Additionally, the following anti-sheep antibodies were used: CD45-FITC and CD44-FITC (Bio-Rad). Cells were stained as per the manufacturers’ instructions, and analysis was completed using an LSR II flow cytometer (BD Biosciences). In this analysis, we also included sheep bone marrow mononuclear cells (MNC) and hBMSC. These cell populations served as controls for antibody performance against a common hBMSC population, and for sheep MNC, where haematopoietic cells (CD45⁺) would be expected. Data were analysed using FlowJo software, version 10 (BD Biosciences).

For evaluation of cell surface marker antigens on the cells separated after MACS, cells were stained with CD146‐FITC (BD Biosciences), CD105‐PE, and CD90‐FITC (Miltenyi Biotec, Germany) based on the protocols for immuofluorescent staining.
To confirm neural differentiation, immunostaining was performed for ß‐tubulin III (Promega cat number: G7121) as a specific antibody against neurons. Briefly, the cells were fixed with 4% PFA in +4°C for 20 min, after that they were permeabilized with 0.01% Triton for 10 min, following by adding blocking solution (5% BSA) for preventing nonspecific binding of antibody. The cells were washed three times with 1X PBS, then they were incubated with primary antibody at concentration of 1:500 for 1 hr in room temperature; after washing primary antibody with 1X PBS, secondary antibody Alexa flour 568 goat antimouse (Abcam cat number #ab175473) was added to the cells and was kept for 45 min in room temperature. The stained cells were observed with Olympus microscope (BX53).