Mitotracker deep red fm

Cells were seeded on coverslips and cultured in 24 well plates for 24 h. After treatment for 24 h, mitochondria were stained with MitoTracker Deep Red FM (Molecular Probes, Carlsbad, USA) according to the manufacturer’s instructions. Cells were fixed with 4% formaldehyde (Beyotime Biotechnology) for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and then blocked with goat serum (Beyotime Biotechnology) in PBS for 30 min. The cells were incubated overnight with primary antibodies at 4 °C, followed by the appropriate secondary antibodies at 37 °C for 1 h. The cells were viewed using a laser-scanning confocal microscope (Zeiss, Germany). All images were analyzed by ImageJ software (MD, USA).

Culturing of HEK293 and COS-7 cells and immunolabeling were essentially as described [42 (link)]. HEK293 and COS-7 cells were transfected using TurboFect (Thermo Scientific). Mitochondria of COS-7 cells were stained with 0.2 μM MitoTracker Deep Red FM (Molecular Probes) in medium at 37°C for 1 h and cells were subsequently fixed with 4% PFA for 7 min.
Primary rat hippocampal neuron cultures for immunofluorescence analyses were prepared, maintained, and transfected (at DIV4) as described previously [16 (link),43 (link),44 (link)]. Fixation was done at DIV6 in 4% (w/v) PFA in PBS pH 7.4 at RT for 4–6 min. Permeabilization and blocking were done with 10% (v/v) horse serum, 5% (w/v) BSA in PBS with 0.2% (v/v) Triton X-100. Phalloidin stainings and antibody incubations were done in the same buffer without Triton X-100 according to [42 (link),44 (link)].

Sperm were isolated from 1 to 2 wild-type and mutant male fish and kept in 100 μl Hank’s saline (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 1.3 mM CaCl₂, 1 mM MgSO₄, and 4.2 mM NaHCO₃) containing 0.5 μM MitoTracker Deep Red FM (Molecular Probes) for >10 min on ice. Sperm (approximately 5,000 sperm/μl) were activated using E3 medium in a 1:4 dilution and placed onto a 10 μm thick chamber slide (Leja counting chamber, SC 10–01–04-B). Sperm motility was imaged 30 s after activation using an Axio Imager.Z2 microscope (Zeiss) and a 10x/0.3 plan-neofluar objective using darkfield. Sperm tracks were analyzed using Fiji with the “manual tracking” plugin (Cordelieres, 2005 ). Sperm that were present in the movie for more than 30 timeframes were tracked for as many frames as possible. Coordinates of the sperm cells were used to calculate average sperm speed and displacement. Sperm displacement was calculated by measuring the distance between the first and the last coordinates (normalized by 100 timeframes).

To visualize mitochondrial membrane potential, cells were treated with 5% CSE for 8 h and afterward incubated with fresh cell culture medium containing MitoTracker Deep Red FM (1:5000; Molecular Probes/Invitrogen, Carlsbad, CA, USA), a polarization-dependent fluorescence staining of mitochondria [38 (link), 39 ], for 15 min in the dark. Cells were washed and fixed with 3% paraformaldehyde containing 0.2% Triton X-100 in PBS. For counterstaining, cells were incubated with Phalloidin-TRITC (1:200) and embedded with Mowiol containing DAPI (1:1000). Fluorescence imaging was conducted with a Zeiss Axio Observer Z1 fluorescence microscope with ApoTome and ZEN imaging software (Carl Zeiss, Goettingen, Germany).

Sperm were squeezed from 2–4 wild-type and mutant male fish and kept in 150 µL Hank’s saline containing 0.5 µM MitoTracker Deep Red FM (Molecular Probes) for >10 min on ice. Un-activated, mature eggs were obtained by squeezing a wild-type female. To prevent activation, eggs were kept in sorting medium (Leibovitz’s medium, 0.5% BSA, pH 9.0) at RT. The eggs were kept in place using a petri dish with cone-shaped agarose molds (1.5% agarose in sorting medium) filled with sorting medium. Imaging was performed with a LSM800 Examiner Z1 upright system (Zeiss) with a 20×/1.0 Plan-Apochromat water dipping objective. Before sperm addition, sorting media was removed and 1 mL of E3 medium was carefully added close to the egg. 5–10 µL of stained sperm was added as close to the egg as possible during image acquisition. The resulting time-lapse movies were analyzed using FIJI.

To investigate the mitochondrial membrane potential, in vitro matured oocytes (triplicates; n = 10–15) were subjected to Mito Tracker Green assay according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Briefly, partially denuded oocytes were stained with 125 nM MitoTracker Green FM for 30 min followed by washing with PBS-PVA. Oocytes mounted on glass slides were examined immediately using inverted epifluorescence microscope and the fluorescence intensity were analyzed using ImageJ software. MitoTracker deep Red FM (Molecular Probes, Eugene, OR, USA) was used to determine mitochondrial distribution pattern in in vitro matured oocytes [45 (link)]. Briefly, oocytes were incubated with 100 nM MitoTracker for 40 min at 38.5 °C then fixed in 4% paraformaldehyde for 15 min at 38.5 °C after washing in PBS-PVA. Oocytes were spotted on glass slides under cover slips and examined using inverted epifluorescence microscope. The mitochondrial distribution patterns were classified either as homogeneous where the mitochondria are dispersed throughout the cytoplasm or as aberrant pattern which shows mitochondrial distribution in cytoplasm either peripheral or semi peripheral pattern located beneath the plasma membrane.

Sperm were isolated from 2 to 4 wild-type and mutant male fish and kept in 150 μl Hank’s saline containing 0.5 μM MitoTracker Deep Red FM (Molecular Probes) for >10 min on ice. Un-activated, mature eggs were then isolated from a wild-type female. To prevent activation, eggs were kept in sorting medium (Leibovitz’s medium, 0.5% BSA, pH 9.0) at room temperature. Eggs were kept in place using a petri dish with cone-shaped agarose molds (1.5% agarose in sorting medium) filled with sorting medium. Imaging was performed with a LSM800 Examiner Z1 upright system (Zeiss) with a 20x/1.0 plan-apochromat water dipping objective. Before sperm addition, sorting media was removed and 1 ml of E3 medium carefully added close to the egg. 3 μl of stained spermatozoa (approximately 150,000—300,000 sperm) was added as close to the egg as possible during image acquisition. The resulting time-lapse movies were analyzed using Fiji. Timestamps were calculated beginning with the addition of sperm to the eggs.