Chicken anti rabbit igg alexafluor 594

Cells were grown on coverslips and then fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with 5% goat serum and 5% bovine serum albumin (BSA) at room temperature, and then incubated with primary antibodies: rabbit polyclonal anti‐OCT4 (1:100; Santa Cruz Biotechnology; sc‐9081), mouse monoclonal anti‐TRA‐1‐81 (1:100; Santa Cruz Biotechnology; sc‐21706), mouse monoclonal anticardiac troponin T (cTnT) (1:100; Abcam; Ab8295) and rabbit polyclonal anti‐α‐actinin (1:100; Abcam; Ab137346) at 4°C overnight. After washing with PBS, the slices were incubated with secondary antibodies: chicken antirabbit IgG Alexa Fluor 594 (1:200; Invitrogen; A21442), chicken antimouse IgG Alexa Fluor 488 (1:200; Invitrogen; A21200), goat antirabbit IgG Alexa Fluor 488 (1:200; Invitrogen; A32731) and goat antimouse IgG Alexa Fluor 594 (1:200; Invitrogen; A21145) at room temperature for 1 hour. Then the slices were rinsed in PBS three times and mounted with DNA‐specific 4′,6‐diamidino‐2‐phenylindole (DAPI; H1200; Vector Lab). Fluorescence was evaluated by a confocal laser scanning microscope (Leica, Wetzlar, Germany).

L-glutamine, penicillin/streptomycin, donkey anti-mouse IgG-Alexa Fluor 488, Chicken anti-rabbit IgG-Alexa Fluor 594, Laemmli sample buffer and Trizol Reagent were purchased from Invitrogen (Carlsbad, CA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 and bovine serum albumin (BSA) were from Sigma (St Louis, MO), and fetal bovine serum (FBS) from Thermo scientific. Rabbit anti α-SMA, anti-CD16 and anti-CD14 bound micromagnetic beads were purchased from Miltenyi Biotec (Auburn, CA), mouse anti-human E-cadherin antibody from BD Biosciences (Mississauga, ON, Canada), and anti-TGF-β₁ monoclonal antibody (mAb) (1D11) from R&D Systems (Minneapolis, MN). Sepasol-RNA I super G (Nacalai tesque), anti-mouse antibodies against CD11b (integrin α_M), CD49d (integrin α₄), CD29 (integrin β₁), CD18 (integrin β₁), CD54 (ICAM-1), and CD106 (VCAM-1) were from BioLegend.