Dylight 488 conjugated goat anti rabbit igg h l

The cells were immobilized in 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X‐100. After blocking with 5% bovine serum albumin, the cells were incubated with anti‐PPARγ antibody (1:250, Abcam) or anti‐α‐smooth muscle actin antibody (1:100, Abcam) overnight at 4°C. The cells were then incubated with 1:1000 DyLight 488‐conjugated goat anti‐rabbit IgG (H+L; Abbkine Inc.), and nuclei were stained with 4,6‐diamidino‐2‐phenylindole (DAPI). Fluorescence images were captured using a fluorescence microscope (Leica DMi8).

IF assays were conducted to identify PASMCs and determine A2aR, SDF-1, and CXCR4 protein expression levels. The pre-treatment procedure was the same as that used for IHC. IF was performed with anti-α-smooth muscle actin (1:1000, ab5694), anti-adenosine receptor A2a (1:200, ab3461), anti-SDF1 (1:200; ab9797), and anti-CXCR4 (1:500, ab124824) antibodies for 12 h in the dark, followed by incubation with 1:1000 DyLight 488-conjugated goat anti-rabbit IgG (H + L) (Abbkine Inc., North Chicago, IL). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a fluorescence microscope (Leica DMi8, Wetzlar, Germany). Quantitative analysis was performed using ImageJ analysis software (National Institutes of Health, Bethesda, MD).