Dm500 icc50

Neuronal apoptosis in the injured region of the spinal cord was analyzed using a TUNEL staining kit (Roche) according to the manufacturer’s instructions. Briefly, the tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, washed in distilled water, treated with proteases, and incubated with the TUNEL reaction mixture. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche). TUNEL-positive cells had a pyknotic nucleus with green fluorescence, indicative of apoptosis. Sections were imaged using a fluorescence microscope (Leica DMI4000B). The TUNEL reaction was also visualized by chromogenic staining with diaminobenzidine (DAB) (Roche). TUNEL-positive cells were stained brownish-yellow, and positive cells were counted. Sections were imaged using a microscope (Leica DM500 ICC50).

Spinal cord tissues were immersed in 4% paraformaldehyde for 24 h, transferred to 70% ethanol, dehydrated through a serial alcohol gradient, embedded in paraffin wax blocks, and serially sectioned into 8 µm-thick segments. Tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in distilled water for HE and Nissl staining. Stained cells were observed using a light microscope (Leica DM500 ICC50).