Goat anti mouse igg h l fitc conjugate

For immunofluorescence 1 × 10⁸ of BF trypanosomes were washed with PBS supplemented with 10 g/l glucose, and spread onto slides coated with polylysine (100 μg/ml; Sigma). The cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), washed with PBS, and permeabilized with 0.1% triton X-100 in PBS. The cells were then washed with 1xPBS-T (0.05% Tween). After blocking with 5.5% FBS, the respective slides were incubated for 1 hr in PBS-T plus 3% BSA with the following primary antibodies: monoclonal anti-v5 (1:200, Invitrogen), anti-enolase (1:400, [36 (link)]) and anti-hexokinase (1:400, a gift from Paul Michels). After washes, the slides were incubated in the dark for 1 hr with the following secondary antibodies: goat anti-rabbit IgG (H + L) Texas Red conjugate (1:400, Life Technologies) or goat anti-mouse IgG (H + L) FITC conjugate (1:400, Sigma). The slides were washed and mounted in Vectashield containing 1.5 μg/ml DAPI (Life Technologies).

Cells in the exponential growth phase were stained with 100 nM red-fluorescent stain MitoTracker Red CMXRos (Thermo Fisher Scientific) for 30 min at 37 °C. After that we followed the protocol published in Ref.^{6 (link)}. Briefly, 1 × 10⁸ of BSF trypanosomes were washed with phosphate-buffered saline supplemented with 10 g/l glucose (PBS-G), and spread onto slides coated with poly-lysin (100 μg/ml). The cells were fixed with 3.7% formaldehyde in PBS, washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS. The cells were then washed with 1xPBS-T (0.05% Tween). After blocking with 5.5% FBS, the respective slides were incubated for 1 h in PBS-T plus 3% BSA with the following primary monoclonal antibody anti-V5 (1:200, Thermo Fisher Scientific). After washes, the slides were incubated in the dark for 1 h with the following secondary antibodies: goat anti-rabbit IgG (H + L) Texas Red conjugate (1:400, Thermo Fisher Scientific) or goat anti-mouse IgG (H + L) FITC conjugate (1:400, Sigma). The slides were washed and mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific).