Testes from 1-day-old males were dissected in phosphate-buffered saline (PBS, pH 7.4), then transferred to 4% polyformaldehyde (PFA) to fix for 30 min at room temperature. The fixed testes were washed with PBST (PBS containing 0.1% Triton X-100) for 30 min and then incubated with primary antibodies, which were diluted in PBST with 0.2% BSA, at 4 °C overnight. After washing for 30 min in PBST, the samples were incubated with the secondary antibody in PBST with 0.2% BSA for 1 h at room temperature followed by washing as above, and mounted in VECTASHIELD® Mounting Medium with DAPI (VectorLabs, Newark, CA, USA). Images were obtained from a Leica SP8 405 Laser confocal microscope (Leica, Wetzlar, Germany) and processed using Adobe Photoshop and ImageJ software (v2.0.0, Wayne Rasband, NIH, USA).
For MitoTracker Red CMXRos (Life Technologies, Shanghai, China) staining, the testes were dissected in PBS and then incubated in the dye solution at a 50 μM concentration for 10 min. The samples were washed three times with PBS and then fixed in 4% formaldehyde for 30 min. The following steps are the same as above. For the phalloidin staining, the TRITC-phalloidin (Solarbio, 1:200) was added together with secondary antibodies to stain the testes.
The primary antibodies used in this study were as follows: rat anti-vasa (Developmental Studies Hybridoma Bank [DSHB, Iowa City, IA, USA], 1:200); mouse anti-α-spectrin (DSHB, 1:400); mouse anti-AXO-49 (Merck, Darmstadt, Germany, 1:5000). The secondary antibodies Alexa Fluor 488/594 conjugated anti-mouse/rat IgG (1:200) were purchased from Life Technologies.
For MitoTracker Red CMXRos (Life Technologies, Shanghai, China) staining, the testes were dissected in PBS and then incubated in the dye solution at a 50 μM concentration for 10 min. The samples were washed three times with PBS and then fixed in 4% formaldehyde for 30 min. The following steps are the same as above. For the phalloidin staining, the TRITC-phalloidin (Solarbio, 1:200) was added together with secondary antibodies to stain the testes.
The primary antibodies used in this study were as follows: rat anti-vasa (Developmental Studies Hybridoma Bank [DSHB, Iowa City, IA, USA], 1:200); mouse anti-α-spectrin (DSHB, 1:400); mouse anti-AXO-49 (Merck, Darmstadt, Germany, 1:5000). The secondary antibodies Alexa Fluor 488/594 conjugated anti-mouse/rat IgG (1:200) were purchased from Life Technologies.
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