Fluorescein isothiocyanate fitc conjugated goat anti rabbit igg h l

Cancer‐associated fibroblasts and NFs were separated and purified by brief exposure to 0.25% trypsin‐EDTA (Invitrogen). Representative images were recorded using a phase‐contrast microscope. Immunocytochemistry was used to verify the identities of CAFs and NFs. The CAFs and NFs cells suspended in BEGM were plated on glass coverslips. The cells were then incubated at 37°C for 24 h and immersed in 4% paraformaldehyde (PFA) for 15 min, washed with PBS and incubated in 10% normal goat serum (Boster) for 40 min to block nonspecific interactions, in the presence or absence of 0.3% Triton X‐100 to permeabilise the cells. The coverslips were immersed in solutions containing primary antibodies (rabbit antihuman) for pan‐cytokeratin (CK; 1:400; Abcam), vimentin (1:200; Abcam), α‐smooth muscle actin (α‐SMA; 1:200; Abcam) and fibroblast activation protein (FAP; 1:250; Abcam) and incubated at 4°C overnight. The cells were then treated with secondary antibodies (Jackson ImmunoResearch), either fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit IgG (H + L; 1:100) or Cy™3‐conjugated goat anti‐rabbit IgG (1:100) for 1 h at 37°C. The cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Boster).

Cells from long-term infected passages were trypsinized, seeded and grown until they reached the confluence on the wells chamber slides (Thermo Fisher Scientific, Waltham, MA, USA). Similarly, non-infected and acutely infected cells were seeded as controls. Fluorescence staining was performed as described by Sarmiento et al.¹¹
with slight adaptations. We used pAb (1:300 in blocking solution) as the primary antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, W. Baltimore Pike, USA) (1:500 in PBST 0.1%) as the secondary antibody. Nuclei were counterstained with DAPI (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Moreover, the cells were viewed using a fluorescence microscope (Nikon, Eclipse E600, Japan).