Alexa fluor 488 chicken anti mouse igg

Human iPSC-derived cardiomyocytes (2 × 10⁴ cells per well) were cultured on precoated multi-well glass-bottom 35-mm dishes (Matsunami, Osaka, Japan) for 8 days. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin (KPL, Gaithersburg, MD, USA) in phosphate-buffered saline, and incubated with primary antibodies for cardiac troponin T (Thermo Fisher Scientific, Waltham, MA, USA) overnight. After washing with phosphate-buffered saline, cells were incubated with the secondary antibody (Alexa Fluor 488 chicken anti-mouse IgG; Invitrogen, Carlsbad, CA, USA) and nuclei were simultaneously counterstained with 4′,6-diamidino-2-phenylindole (Molecular Probes, Carlsbad, CA, USA) for 1 h. Images were acquired with a confocal microscope (TCS SP8; Leica Microsystems, Tokyo, Japan).

For immunofluorescent staining, pHEPs and Kupffer cells isolated from WT and KO male/female mice were harvested and deposited in a monolayer onto a defined area of a slide by cytospin centrifugation (Cell spin; Hanil Scientific, Gimpo, Korea). Isolated cells were fixed in and permeabilized with cold acetone and methanol, respectively. These cells washed with TBS were incubated with blocking solution (X9090; Dako) for 30 min, and were treated with primary antibody, anti-Fpr2 (ab203129; Abcam) for at 4 °C overnight. After being washed with TBS, they were incubated with fluorescein-labeled secondary antibody, Alexa Fluor 568 goat anti-rabbit IgG (diluted 1:100; Invitrogen) for 30 min at room temperature. For double immunofluorescent staining, pHEPs stained for Fpr2 were further incubated with blocking solution for 10 min and then treated with secondary primary antibody, anti-albumin (sc-69873; Santacruz) for 2 h at room temperature. After being washed, their pHEPs were incubated with fluorescein-labeled secondary antibody, Alexa Fluor 488 chicken anti-mouse IgG (diluted 1:100; Invitrogen) for 30 min at room temperature. Slides were mounted on slides antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, VectaShield, Burlingame, CA, USA). Slides were viewed with a Zeiss LSM 800 confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA).