Alexa fluor 568

Free-floating sections were incubated for 1 hr in a blocking solution (PBS containing 0.1% Triton X-100 and 5% BSA). The tissue was then incubated overnight at room temperature in primary antibodies: goat anti-choline acetyl transferase [ChAT; 1:100; Millipore, Burlington, MA, USA] alone for co-labelling with GCaMP6, or rabbit anti-tryptophan hydroxylase [TPH; 1:500; Sigma-Aldrich, St Louis, MO, USA] alone for co-labelling with GCaMP6.
Slices were washed in PBS (6 × 5  min) and then incubated in a blocking solution to which secondary antibodies were added; donkey anti-rabbit Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6, or donkey anti-goat Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA; RTN) for co-labelling with GCaMP6 in RTN tissue or donkey anti-goat Alexa Fluor 568 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6 in pF_L tissue. The tissue was then incubated 2–4 hr at room temperature. Tissue was washed in PBS (6 × 5  min). Slices were mounted on polysine adhesion slides and were coverslipped with Vectashield Antifade Mounting Medium with DAPI (Vectorlabs, Burlingame, CA, United States).

The paraffin section was dewaxed and heated in a 95 °C water bath for 20 min in sodium citrate antigen repair solution (Beyotime Biotechnology) and naturally cooled to room temperature (cell samples were fixed with 4% paraformaldehyde for 15 min). Sections or cells were blocked for 2 h at room temperature and incubated at 4 °C for 20 h with primary antibodies against Drp1 (1:200, Abcam, UK) and mitochondrial preprotein translocases of the outer membrane (Tom20, 1:100, Santa Cruz, CA, USA). After washing, they were incubated with Alexa Fluor 568 (1:1000, Jackson, PA, USA) and Alexa Fluor 488 (1:1000, Jackson, PA, USA) secondary antibodies for 1 h at room temperature in the dark. After washing, they were incubated with DAPI (Beyotime Biotechnology) for 5 min. Sections or cells were imaged with a confocal microscope (Olympus Fluoview FV1000, Tokyo, Japan). More than 5 pictures were taken for each sample. Cell samples were taken with more than 30 cells in each group. In each image, the ImageJ plug-in Coloc 2 was used to detect the Manders co-localization Coefficient (MCC) and to evaluate the co-localization of mitochondria and Drp1.

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)].

Immunofluorescence experiments were carried out following our previously reported protocols [21 (link)]. Briefly, cells growing on the glass slide were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 diluted in PBS for 10 min at room temperature. To block unspecific epitopes, cells were incubated with PBS containing 1% BSA and 0.1% Tween-20 for 1 h. Cells were then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-OCT4 (5 μg/ml, Abcam, Nanchang, China), mouse anti-SSEA-4 (15 μg/ml, Abcam), rabbit anti-Nanog (1:200, Abcam), rabbit anti-Ki67 (1:100, Abcam), and mouse anti-PCNA (5 μg/ml, Abcam). After that, cells were incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were counterstained with DAPI (Thermo Fisher).