Ts 1000 microscope

Immunofluorescence was conducted using an anti-PRDXs-SO₃ antibody to visualize PRDXs-SO₃ expression in control and treated samples. Briefly, mouse spermatozoa were air-dried and fixed with 3.7% paraformaldehyde for 30 min at 4 °C^{50 (link)}. Air-dried slides were washed with Dulbecco’s phosphate-buffered saline (DPBS) containing 0.1% Tween 20 (PBS-T) and blocked for 1 h in blocking solution (5% BSA in PBS-T) at 4 °C. After blocking, slides were incubated with rabbit polyclonal primary antibodies against peroxiredoxins-SO₃ (Abcam) (1:100 in blocking solution), or lectin PNA 34 and 35 conjugated to Alexa Fluor 647 (Molecular Probes) (1:100 in blocking solution) overnight at 4 °C. Next, slides were washed with PBS-T and incubated at room temperature for 2 h with a fluorescein isothiocyanate-conjugated goat polyclonal rabbit IgG secondary antibody (Abcam) (1:100 in blocking solution). Finally, slides were counterstained with Hoechst 33342, mounted with antifade reagent and imaged using a Nikon TS-1000 microscope and NIS Elements image software (Nikon, Tokyo, Japan).

After 1 week of culture, the germ cells were fixed for 30 min at 37 °C with 4% paraformaldehyde and permeabilized for 10 min at room temperature in 0.1% Triton X-100 with DPBS. For blocking, the cells were incubated with 5% (w/v) bovine serum albumin (BSA) for 1 h, and then with a mouse anti-human promyelocytic leukemia zinc finger (PLZF, 1:200; EDM Millipore, Billerica, MA, USA), rabbit anti-human glial-derived neurotrophic factor family receptor alpha 1 (GFRα1, 1:200; Abcam, Cambridge, UK), rabbit anti-human DDX4 (VASA, 1:200; Abcam), and goat anti-mouse c-Kit (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA), and rabbit anti-Ki67 (1:200; Abcam) primary antibody at 4 °C for 12 h. After washing 3 times with DPBS, the cells were incubated with an Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 568-conjugated donkey anti-rabbit IgG, and Alexa Fluor 568-conjugated donkey anti-goat IgG at room temperature for 1 hr. After incubation with the secondary antibody, the cells were washed with DPBS. VectaShield mounting media including DAPI was used to mount the cells. The labeled cells were analyzed using a Nikon TS-1000 microscope with NIS Elements imaging software (Nikon, Chiyoda-ku, Tokyo, Japan).

The subcellular localization of proteins is very important because it can be readily used to obtain information about their potential function. To evaluate the subcellular localization of APN in the freshly collected epididymal mouse spermatozoa, an immunofluorescence assay was performed using the CD13 antibody. Briefly, the air-dried spermatozoa placed on glass slides were fixed with 3.7% paraformaldehyde for 30 min at 4°C [22 (link)]. After washing with Dulbecco's phosphate-buffered saline (DPBS) containing 0.1% Tween 20 (PBS-T) and blocking for 1 h in the blocking solution (5% BSA in PBS-T) at 4°C, the slides were incubated with diluted rabbit polyclonal primary antibody for APN (1:100; Abcam) in blocking solution, and diluted lectin PNA 34 and 35 antibodies (1:100) conjugated with Alexa Fluor 647 (Molecular Probes) in blocking solution overnight at 4°C. After washing, the slides were incubated for 2 h at room temperature (RT) with diluted fluorescein isothiocyanate-conjugated goat polyclonal secondary antibody to rabbit IgG (1:100; Abcam) in blocking solution. After applying the Hoechst 33342 and antifade reagents, the samples were observed with a Nikon TS-1000 microscope using the NIS-Elements imaging software (Nikon, Tokyo, Japan).