Dylight 488 affinipure goat anti mouse igg

Hippocampal NSCs were purified after density centrifugation and cultured in 12-well plates for 3 days according to the following study. Samples were fixed with 4% paraformaldehyde (PFA), blocked by goat serum (diluted into PBST; 0.01 mol/L PBS, 0.02% Triton-100), and immunostained with mouse anti-Nestin (1 : 500; Merck Millipore, Billerica, MA, USA) for 24 hours in the dark at 4°C. Sections were then incubated with secondary antibodies (Dy-Light 488 AffiniPure goat anti-mouse IgG (1 : 250, Jackson, PA, USA) for 1 hour in the dark at 37°C. Nuclei were counterstained with Hoechst 33342 (Beyotime Institute of Biotechnology, China). Sections were observed under a fluorescence microscope. Pictures were captured under fluorescence microscope with 10x objective.

Surface expression of CXCR7, Flk-1, Flt-1, vWF, VE-cadherin, and CD31 was evaluated by flow cytometric analysis. Cells were harvested with PBS containing 5 mM EDTA and immediately neutralized in FACS buffer (α-MEM containing 1% BSA and 0.025% NaN₃). After extensive washing with FACS buffer, cells (10⁵ cells) were incubated with 1 µg/ml of the primary antibodies, including CXCR7 (MAB42273, R&D Systems, 1:100 dilution), Flk-1 (Avas12a1, Novus, 1:100 dilution), Flt-1 (MAB321, R&D System, 1:100 dilution), vWF (ab8822, Abcam, 1:100 dilution), VE-cadherin (FAB9381P, R&D System, 1:100 dilution), and CD31 (FAB806G, R&D Systems, 1:100 dilution) by shaking for 1 h at 4 °C. After extensive washing with FACS buffer, cells were incubated with DyLight 649 AffiniPure goat anti-rabbit IgG or DyLight 488 AffiniPure goat anti-mouse IgG (115-495-209 or 111-545-144, Jackson Immunoresearch, 1:100 dilutions) by shaking for 1 h at 4 °C. Cells were then washed with FACS buffer five to six times, and fixed in PBS containing 1% paraformaldehyde. Expression levels were measured on a FACScalibur instrument and FACSDiva 6.0 software (BD Bioscience).

Surface expression of CXCR7 or CXCR4 was evaluated by flow cytometric analysis. Cells were harvested with phosphate-buffered saline (PBS) containing 5 mM EDTA and immediately neutralized in fluorescence-activated cell sorting (FACS) buffer (α-MEM containing 1% BSA and 0.025% NaN₃). After extensive washing with FACS buffer, cells (10⁵ cells) were incubated with 1 μg/ml of the primary antibodies, including CXCR7 and CXCR4 (R&D Systems), in a 1:100 dilution by shaking for 1 h at 4°C. After extensive washing with FACS buffer, cells were incubated with DyLight 649 AffiniPure goat anti-rabbit IgG or DyLight 488 AffiniPure goat anti-mouse IgG (Jackson Immunoresearch, 1:100 dilutions) by shaking for 1 h at 4°C. Cells were then washed with FACS buffer five to six times and fixed in PBS containing 1% paraformaldehyde. Besides, cell suspension derived from synovial tissue digestion was separated into different tubes and stained with FITC-CD11b or PE-Foxp3 to a determine the frequency of CD11b⁺ macrophages and Foxp3⁺ Treg cells in synovial tissues. Expression levels were measured on a FACScalibur instrument and with FACSDiva 6.0 software (BD Bioscience).