Alexa fluor 488 goat anti rabbit sfx kit

Cells were stained with the following antibodies obtained from BD Biosciences (Franklin Lakes, NJ, USA), eBioscience, or BioLegend (San Diego, CA, USA): anti-CD4 (BioLegend, 100451), anti-CXCR3 (eBioscience, 12-1831-82), anti-CCR7 (eBioscience, 12-1971-82), anti-CD44 (eBioscience, 12-0441-83), anti-CD62L (BioLegend, 104406), anti-CD11c (BD Bioscience, 553801), anti-MHCII (BioLegend, 107631), anti-CD80 (BD Biosciences, 553769), anti-CD86 (BD Biosciences, 553692), anti-B220 (eBioscience, 12-0452-83), anti-CD8 (BD Bioscience, 553032), anti-CD103 (BD Bioscience, 557495), anti-CD45 (BioLegend, 103132), and anti-CD11b (BioLegend, 101263). For Th1 and Treg analyses, cells were stained for surface markers, permeabilized with the Intracellular Fixation and Permeabilization Buffer Set (eBioscience, 88-8824-00), and then stained with anti-IFN-γ (BioLegend, 505825) and anti-FOXP3 (eBioscience, 17-5773-82) antibodies. The following antibodies were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA): anti-Ezh2 (#5246), anti-Runx1 (#4336), and anti-H3K27me3 (#9733). The Alexa Fluor™ 488 Goat Anti-Rabbit SFX Kit from Invitrogen (Carlsbad, CA, USA) was used as a secondary antibody. Multicolor flow cytometric analysis was performed using a CytoFLEX LX (Beckman Coulter, Indianapolis, IN, USA).

C2C12 cells grown in a covered glass-bottom dish (SPL, Yeoju, Republic of Korea) were treated with differentiation medium and stained for CTNNA2 protein following 0 and 6 days of incubation. Briefly, the cells were rinsed with PBS and fixed with 4% formaldehyde. Following permeabilization with 0.2% Triton X-100 (Sigma-Aldrich, MO, USA) for 5 min, the cells were incubated overnight with anti-CTNNA2 antibody (1:50; Abcam, MA, USA) at 4°C in a humid environment. Secondary antibody (1:100; Alexa Fluor 488 goat anti-rabbit SFX kit; Invitrogen) was then applied for 1 h at room temperature. Next, the samples were rinsed with PBS after which the nuclei were counterstained with 4’ 6’-diamino-2-phenylindole (DAPI; Sigma-Aldrich). Finally, pictures were taken using a fluorescent microscope equipped with a digital camera (Nikon, NY, USA).