Alexafluor 594 donkey anti goat 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).

K562 cells were subcutaneously injected into the left flank of NOG mice, while K562-CD19 cells were subcutaneously injected into the right cavity of the same mouse. Ten days after tumor cell injection, GMCs were injected into the cardiac chamber. Five days after GMC injection, mice were sacrificed, and paraffin-embedded tissues were prepared. Immunohistochemistry was performed with an anti-CD3 antibody (ab109531; Abcam, Cambridge, MA) and Alexa Fluor 488 chicken anti-rabbit immunoglobulin G (IgG) (Abcam) to detect injected GMCs. We also performed immunohistochemistry with an anti-IFN-γ antibody (AF-285-NA; R&D Systems, Minneapolis, MN) and Alexa Fluor 594 donkey anti-goat IgG (Abcam) to detect activated GMCs. Nuclei were stained with DAPI (SlowFade Goldantifade reagent with DAPI; Thermo Fisher Scientific). Images were obtained with a fluorescence microscope (VS120-L100; Olympus, Tokyo, Japan).