Anti mouse igg fitc

Cells were seeded onto glass coverslips in 12-well plates. The subcellular localization of MAV2054 was analysed using a confocal microscope (LSM510 META; Carl Zeiss, Heidelberg, Germany). The cells were incubated with Mitotracker Red (Molecular Probes). After washing, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilised with 0.1% Triton X-100 in PBS for 10 min, blocked with PBS containing 3% bovine serum albumin for 1 h, stained with primary anti-MAV2054 antibody for 1 h, and then stained with secondary antibody (anti-mouse IgG FITC; DAKO, Glostrup, Denmark) for 1 h. DAPI staining was conducted for 10 min. Cells were washed with PBS between each step. The cytosol localization of MAV2054 was analysed using a confocal microscope. The cells were fixed, permeabilised, and stained with an anti-MAV2054 antibody followed by an anti-mouse IgG FITC (DAKO). After DAPI staining with Texas Red®-X phalloidin (Molecular Probes) for 20 min, the cells were imaged under a confocal microscope (Carl Zeiss).

Minor melanoma tissue samples were snap-frozen with isopentane in liquid nitrogen. Fresh frozen cancerous tissue cryostat sections [6–8 μm, freezing media (Bio-Optika, Milano, Slee Cryostat mnt (Auroscience)] of melanomas were fixed in 4% paraformaldehyde (PFA) PBS for 15 min. Three percent BSA PBS was used for blocking, before the monoclonal antibodies specific to tumor-associated disialylated glycosphingolipid antigens (Calbiochem, Abcam) were added for an overnight incubation at 4°C. Indirect immunofluorescence with FITC-labeled second antibodies (anti mouse IgG FITC from DAKO), or biotinylated rabbit anti mouse IgG (Fab')2 (1:100) and Streptavidin FITC (Vector Laboratories, Burlingame, CA, USA) with propidium-iodide (nuclear staining) was used. Chamber slides (Nunc Lab Tech) were used in certain cases to culture cancerous cells and IF label them in situ. Indirect immunofluorescence (FITC) labeling was detected in confocal laser microscopy (Nikon Eclipse E600, Nikon Model C1-Lu3, Tokyo, Japan) or conventional IF microscopy.

Cells were cultured on round coverslips inside 24 well plates (Orange Scientific, Braine-l’Alleud, Belgium) for 24 h. Then cells were washed, fixed, and permeabilised with 0.1% TritonX100 and blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA). Cells were subjected to immunofluorescence staining with anti-sLe^X/A (1:50; HECA-452 clone; Biolegend, San Diego, CA, USA) and anti-CK5/6 (1:200; Cell Marque, Rocklin, CA, USA). Cells were incubated with anti-rat IgM-Fluorescein isothiocyanate (FITC) (1:50) (BD Pharmingen, San Diego, CA, USA) or anti-mouse IgG-FITC (1:100) (Dako, Santa Clara, CA, USA) secondary antibodies. Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (1μg/mL, ThermoFisher, Waltham, MA, USA) for 10 min, washed, mounted with montage medium Mowiol+1,4-diazabicyclo [2.2.2]octan, and analysed. Fluorescence intensities from five randomly selected microscopic fields of cells were quantitatively analysed with ImageJ software and using the corrected total cell fluorescence (CTCF) formula: CTCF = Integrated density – (Area of selected cell x Mean fluorescence of background readings).