Alexafluor 647 donkey anti mouse igg

The following primary antibodies were used: α-smooth muscle actin (αSMA), SM22α, CD31, CD34, von Willebrand factor (vWF), GFP, cardiac troponin T, anti-vascular endothelial growth factor receptor-2 (VEGFR-2), transforming growth factor β1 (TGF-β1), TGF-β receptor 1 (TGF-β R1), TGF-β receptor 2 (TGF-β R2), caspase-3, caspase-9, vinculin, mouse IgG polyclonal isotype control, rabbit IgG polyclonal isotype control, and goat IgG polyclonal isotype control (Abcam, Cambridge, MA). Fluorescent-conjugated secondary antibodies included: AlexaFluor^®488 donkey anti-mouse IgG, AlexaFluor^®594 donkey anti-rabbit IgG, AlexaFluor^®594 donkey anti-mouse IgG, AlexaFluor^®594 donkey anti-goat IgG, AlexaFluor^®488 donkey anti-rabbit IgG, AlexaFluor^®488 donkey anti-goat IgG, AlexaFluor^®594 donkey anti-rabbit IgG, and AlexaFluor^®647 donkey anti-mouse IgG (Abcam); horseradish peroxidase-conjugated secondary antibody (N-Histofine^®, NICHIREI Biosciences Inc., Tokyo, Japan); 4′ 6-diamidino-2-phenylindole (DAPI, NucBlue^® Fixed Cell ReadyProbes^® Reagent, ThermoFisher Scientific, Waltham, MA).

For labeling, decidual tissue sections were incubated with primary antibodies: mouse monoclonal anti-SGK1 antibodies (1:50, SC-28338, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-CD68 antibodies (1:500, 76437, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The slides of THP-1 cells before and after phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, P1585) pretreatment were incubated with anti-CD68 antibodies at 4°C overnight. Then, the sections of human decidual tissue and THP-1 macrophages were incubated with Alexa Fluor® 488 Donkey Anti-Rabbit IgG (1:400, ab150073, Abcam, Cambridge, UK) and Alexa Fluor® 647 Donkey Anti-Mouse IgG (1:400, ab150107, Abcam, UK) secondary antibodies at 37°C for 1 h in the dark. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, C1005, Beyotime Biotechnology, Shanghai, China) and washed using PBS (3 × 5 min). The slices were photographed and results were recorded using a laser confocal microscope (LSM 710, Zeiss, Wetzlar, Germany).