Alexa fluor 568 conjugated goat anti rabbit igg antibody

Isolated cells were suspended in McCoy's 5A medium containing 5% FBS and 50 μg ml⁻¹ gentamicin and seeded in 8 well-chamber slides at the density of 250 000 cells per well and incubated overnight. The cells were washed and fixed with 4% paraformaldehyde and permeabilized with 0.1% triton-X 100 (Thermo Fisher Scientific, Waltham, MA). Nonspecific sites were blocked by incubating the cells with 1% bovine serum albumin (BSA) solution for 1 h. The cells then were stained with 1 : 500 diluted rabbit anti-pro-SP-C antibody (Sigma, St. Louis, MO) overnight at 4 °C, washed, and incubated in 1 : 200 diluted Alexa Fluor 568-conjugated goat anti-rabbit IgG antibody (Abcam, Waltham, MA) for 1 h at room temperature. After washing with DPBS, the cells were stained with 1 : 2000 diluted Hoechst 33342 dye (nuclear stain, Thermo Fisher Scientific, Waltham, MA). The immunostained cells were mounted using ProLong Gold anti-fade reagent (Thermo Fisher Scientific, Waltham, MA). The imaging was done by Leica SP8 confocal microscope at 40× objective. Photomicrographs were taken from random regions.