Slides were extensively washed in PBS and cover-slipped with DAPI (Sigma, St Louis, MO) in Fluoromont G mounting media (Southern Biotech, Birmingham, AL).
Next, the slides were extensively washed with PBS and immunostained for 30 minutes at RT with a 1∶250 dilution of biotinylated goat anti mouse IgG (Southern Biotech).
Slides were extensively washed and then incubated for 1 hour at 37°C with anti-DIG fluorescein isothiocynate (FITC) conjugated antibody, washed again, drained and counterstained with 4'-6'Diamidino-2-phenylindole (DAPI), before the addition of ProLong® Gold antifade reagent (Invitrogen).
Finally, the slides were extensively washed and mounted on a coverslip with the DAPI Mounting Medium (OLink Bioscences) to stain the nuclei.
After each antibody incubation, the slides were extensively washed twice with PBS containing 0.05% Tween-20, and once with PBS.
Between all incubation steps, slides were extensively washed with tris-buffered saline containing 0.02% v/v Tween®-20 detergent (TBST) (Thermo Fisher Scientific).
After the slides were extensively washed with PBS, mounted with 90% glycerol, and examined under an Axioskope microscope (Carl Zeiss, Oberkochen, Germany).
After incubation, the slides were extensively washed with PBS and incubated with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), (1 1,000), (Sigma-Aldrich Srl) and donkey anti-mouse IgG-FITC (1 300; purchased from Alexa Fluor, Invitrogen).
Slides were washed extensively in PBS and mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
The slides were washed extensively with PBS and mounted with Fluoromount-G (SouthernBiotech).
