Vectashied mounting medium

Nuclei and kinetoplasts were visualized by using the fluorescent dye 4’,6-diamidino-2-phenylindole (DAPI) on BF trypanosomes after fixation. 50 μl of cells at 2 × 10⁶ cells/ml were spread onto a glass microscope slide, left to dry in a fume hood and fixed in 4% paraformaldehyde in fresh PBS for 10 min at room temperature. The slides were washed with 1 × PBS. The PBS was gently removed by inclining the slide on a soft tissue and a drop of Vectashied^® mounting medium containing DAPI dihydrochloride (Vector Laboratories, USA) was placed on the slide before covering with a cover slip. The slide was then viewed under UV light on a Zeiss Axioskop 2 fluorescent microscope (Carl Zeiss Microscopy, USA). Five hundred cells were recorded for each sample, and scored for DNA configuration following these groups: 1N1K, 1N2K, 2N2K (Early) and 2N2K (Late) (N, nucleus; K, kinetoplast). The effect of test compounds on DNA configuration was determined at 0, 8 and 24 h; untreated cultures served as control.

Immunostaining of mesangial cells were carried out at day 28^th of culture. as described by us previously74 (link). Briefly, fixed and permeabilized cells were pre-incubated with PBS containing 1% BSA for 1 h at 37 °C followed by overningt incubation (at 4 °C) with mouse anti-vimentin antibodies (Invitrogen). In addition, mouse anticytokeratin (pan), clone AE1/AE3 antibodies (Invitrogen) was used as epithelial cells marker or only PBS with 1% BSA was used in place of primary antibodies as negative control. After PBS washing, cells were incubated for 1 h with rabbit anti-mouse IgG FITC conjugate (diluted 1:50 in PBS containing 1% BSA), washed and mounted in VECTASHIED mounting medium (Vector Laboratories, Burlingame, CA) and observed under Confocal microscope (OLYMPUS, FV10i).