Lsm900 confocal microscope system

The testes of mice were fixed with 4% paraformaldehyde (PFA) overnight at 4 °C. Paraffin embedding and sectioning were then conducted using the usual procedures. The slices were dewaxed and rehydrated, immersed in 0.01% sodium citrate buffer (pH 6.0, Solarbio, Beijing, China), and boiled in water for 20 min. Next, the slices were permeated with 0.2% TrionX-100 at room temperature for 10 min. After being washed with phosphate-buffered saline (PBS), the samples were blocked with 5% goat serum at 37 °C for 30 min. Further, the sample was incubated with the primary antibodies at 4 °C overnight. The secondary antibodies conjugated to fluorescein isothiocyanate (FITC, 1:200; Invitrogen, USA) or tetramethylrhodamine isothiocyanate Fluor (TRITC, 1:200; Invitrogen, USA) were used to test the primary antibody. 4′,6-diamino-2-phenylindole (DAPI; Abcam, Cambridge, UK) was used to stain the nuclei. The samples were observed under the LSM900 confocal microscope system (Carl Zeiss AG, Jena, Germany), and the images were captured. The following antibodies were used: anti-γH2AX (1:200; Abcam, Cambridge, UK), anti-DEAD-box helicase 4 (DDX4) (1:200; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (PCNA) (1:50; Santa, Texas, USA), anti-GSS (1:200; Affinity Biosciences, Jiangsu, China) antibodies.

Rats were anesthetized with 2% pentobarbital sodium, and the blood was removed by transcardial perfusion with PBS. A 5-mm segment of spinal cord tissue surrounding the injury site was harvested for histological analysis after perfusion with 4% paraformaldehyde. The spinal cord segment was then embedded in OCT embedding agent, and 16 μm sagittal sections were obtained using a microtome (RM2235, Leica, Germany). The sections were blocked in 10% donkey serum containing 0.3% Triton X-100 (SL050 and T8200, Solarbio, China) at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 °C. The primary antibodies included mouse anti-NF200 (1:500, ab213128, Abcam, USA), rabbit anti-GFAP (1:200, 16825-1-AP, Proteintech, China), mouse anti-hBcl2 (1:100, NBP2-15200, Novus, USA), and rabbit anti-cleaved caspase3 (1:100, ab32042, Abcam, USA). Then, the sections were incubated with secondary antibodies at room-temperature for 1 h. The secondary antibodies used were donkey anti-mouse Alexa Fluor 555, donkey anti-rabbit Alexa Fluor 488, and donkey anti-mouse Alexa Fluor 488, donkey anti- rabbit Alexa Fluor 555 (1:500, Termo Fisher Scientific, USA). Finally, the sections were stained with DAPI to label the nuclei. The images of the sections were acquired using a Zeiss LSM 900 confocal microscope system and a Zeiss Axio Scope A1 fluorescence microscope.