Alexa fluor 594 donkey anti mouse igg h l

Cells were fixed in 4% paraformaldehyde/PBS (Nacalai tesque) for 15 min, permeabilized with 0.25% Triton X-100 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) in PBS for 10 min and blocked with 1% bovine serum albumin (BSA, A9647, Sigma-Aldrich) in PBS for 1hr at room temperature (RT). Cells were incubated with primary antibodies (1:500 dilution in 1% BSA) overnight at 4°C and with secondary antibodies (1:1000 dilution in 1% BSA) for 1 h at RT. Primary antibodies used were: anti-NeuN (Cat# MAB377, Millipore, Tokyo, Japan, RRID:AB_2298772), anti-GFAP (Cat# ab53554, Abcam, Tokyo, Japan, RRID:AB_880202), and anti-Iba1 (Cat# 019-19741, FUJIFILM Wako Shibayagi Corp, Shibukawa, Japan; RRID:AB_839504). Secondary antibodies used were: Alexa Fluor 594 donkey anti-mouse IgG H&L (Cat# ab150108, Abcam, RRID:AB_2732073), Alexa Fluor 488 chicken anti-goat IgG H&L (Cat# A-21467, Molecular Probes, RRID:AB_141893), and Alexa Fluor 594 donkey anti-rabbit IgG (H+L) (Cat# A-21207, Thermo Fisher Scientific, RRID:AB_141637). Nuclei were stained with DAPI (sc-3598, Santa Cruz Biotechnology, Dallas, TX, USA). Images were obtained with an inverted microscope (IX71, Olympus, Tokyo, Japan) with a 20× objective (UApo/340, N.A. 0.75, Olympus) and a cooled-CCD camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan).

Ginsenoside Rg1 was obtained from Shanghai Yuan Ye Biological Technology Co., Ltd(B21057). The endothelial cell medium (ECM) was acquired from Science ll (Carlsbad, CA, USA). Green streptomycin and Pancreatic enzymes were purchased from Hyclone (SH30042.01). CD31 antibody, VWF antibody, Alexa Fluor 488 donkey anti-rabbit IgG (H+L), and Alexa Fluor 594 donkey anti-mouse IgG(H+L) were purchased from Abcam (Cambridge, UK). BSA, DMSO, triphenyl tetrazolium chloride (TTC), and dilution buffer were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). PBS was purchased from Nanjing Sheng Xing Co. Paraformaldehyde, TritonX-100, and Toluidine blue were obtained from Aladdin (Los Angeles, CA, USA). MTT was purchased from Beyotime Co. (Shanghai, China). SYBR Green I, an RT-PCR kit, and RNAiso Plus were purchased from Takara (Kyoto, Japan).