Cytm3 conjugated goat anti rabbit igg

Harvested CAFs and NFs were seeded onto glass coverslips, incubated for 24 h, rinsed in PBS, and then fixed with 4% paraformaldehyde for 15 min. This was followed by incubation in 5% bovine serum albumin containing 10% normal goat serum (Boster, Wuhan, China) (in the presence or absence of 0.3% Triton X-100 to permeabilize the cells) at room temperature for 40 min to block nonspecific interactions. The cells were then incubated with rabbit anti-mouse primary antibodies of pan-cytokeratin (CK) (1:200; Epitomics, Burlingame, CA, USA), vimentin (1:200; Epitomics), α-smooth muscle actin (α-SMA) (1:200; Epitomics), and fibroblast activation protein (Fap) (1:250; Abcam, Cambridge, UK) at 4°C overnight. After being washed, the cells were incubated with secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H+L) (1:100) or Cy^TM3-conjugated goat anti-rabbit IgG (1:200) in the dark for 1 h at 37°C. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Boster). Ten randomly selected microscopic fields (×200) of vimentin staining were analyzed to calculate the purities of the CAFs and NFs. Epithelial cells from the skins of the nude mice were primary cultured and served as positive control in pan-CK staining.