Lms 700 confocal microscope

Trophozoites were grown on coverslips, fixed with 4% paraformaldehyde at 37 °C for 1 h, permeabilized with 0.5% Triton X-100, and blocked with 10% fetal bovine serum in PBS. Then, cells were incubated at 4 °C overnight (ON) with either α-EhVps22 (1:100), α-EhVps25 (1:100), α-EhVps36 (1:100), α-EhVps23 (1:50), or α-EhVps20 (1:50) antibodies. After extensive washing, samples were incubated for 30 min at 37 °C with α-mouse pacific blue-labeled, α-rat TRITC-labeled, or α-rabbit FITC-labeled secondary antibodies (1:100). Nuclei were stained with 40′6-Diamidino-2- Phenylindole (DAPI). Fluorescence was preserved using VECTASHIELD antifade reagent (Vector), examined through a Carl Zeiss LMS 700 confocal microscope in laser sections of 0.5 µm, and processed with ZEN 2009 Light Edition Software (Zeiss, Dublin, CA, USA). We considered at least 25 confocal images using the ImageJ 1.45v software and JACoP plugin to evaluate the fluorescence intensity and co-localization between proteins.

Skin cell suspensions were obtained by Liberase digestion as mentioned above. Cell suspensions were diluted in RPMI and deposited in microplates containing poly-L-lysine-coated coverslips (Corning). Cells were incubated overnight at 4°C and fixed with 4% paraformaldehyde. Cells were then incubated with rat anti-mouse MHC-II (clone 2G9, BD Bioscience) and rabbit anti-clathrin heavy chain (Abcam), followed by AF647 goat anti-rabbit IgG (Invitrogen) and AF-555 goat anti-rat IgG (Invitrogen). Finally, coverslips were mounted on microscope slides using ProLong Gold with DAPI (Life Technologies). Cells were visualized with a LMS 700 confocal microscope (Zeiss) and pictures were edited using Zen software (Zeiss).

SKOV-3 and OVCAR-3 cells adhered to coverslips were subjected to different treatment conditions. Upon completion, the cells were fixed with 4% paraformaldehyde for 1 h at 37 °C. Cells were rinsed 3× with filtered 1× PBS and permeabilized with a 0.2% solution of Triton X-100 in 1× PBS for 15 min at room temperature with stirring. Cells were rinsed thrice with filtered 1× PBS and blocked with 10% FBS for 1 h at 37 °C. The primary antibody (Sigma-Aldrich HPA028417) was added at a 1:100 dilution in 1× PBS and incubated overnight at 4 °C in a humid chamber. On the next day, cells were rinsed 3× with 1× PBS. TRITC goat anti-rabbit (IgG) secondary antibody (ab50598, Abcam) was added to OVCAR-3 cells and incubated for 1 h at 37 °C under protection from light. For the SKOV-3 assays, a different secondary antibody was employed: Alexa Fluor 647 donkey anti-rabbit (IgG) secondary antibody (1:100 dilution) (711-605-152, Jackson InmunoResearch Laboratories), this with the objective to improve the signal of the protein. Vectashield with DAPI (Cat. No. H-1200) was added, and the coverslip was mounted on a slide. The assembled samples were stored at − 20 °C protected from light until observation under a Carl Zeiss LMS 700 confocal microscope. ZEN 2012 software was used to analyze the samples (Carl Zeiss).