Immunofluorescence of SUMO and Surface Proteins in Trypanosoma brucei

BSF cells were collected by centrifugation (1000 x g for 10 min), washed with TDB supplemented with glucose 20 mM and fixed with 4% paraformaldehyde (PFA) in PBS for 1 h. Parasites were allowed to bind to poly-L-lysine coated glass coverslips for 30 min and then incubated with 25 mM NH₄Cl for 15 min. Permeabilization and blocking were performed with 3% bovine serum albumin (BSA), 0.5% saponin and 5% normal goat serum in PBS for 1 h. Mouse anti-TbSUMO (1:500) [14 (link)], mouse anti-VSG221 (1:200), rabbit anti-VSG221 (1:500), mouse anti-EP FITC (1:500) (Cederlane Laboratories, Burlington, Canada) or anti-PAD1 [58 (link)] were used as primary antibodies. After washing with PBS, coverslips were incubated for 1 h with secondary antibodies diluted 1:1000 in 1% BSA:PBS (polyclonal goat anti-rabbit Alexa Fluor 568 or polyclonal goat anti-mouse Alexa Fluor 488 (Jackson)). Finally, coverslips were extensively washed and mounted using FluorSave reagent (Merck, Darmstadt, Germany). Nucleus and kinetoplast were visualized with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Samples were analyzed with an Eclipse 80i microscope (Nikon, Shinagawa, Japan). For SUMO visualization images were acquired as 3D z-stacks, deconvoluted and projected into 2D using a maximum intensity projection, as previously done in López Farfán et al., 2014 [14 (link)].