Processing of Spinal Cord Tissue for NeuN, GFAP and GAP-43 Immunostaining and Quantification

While collecting the sciatic nerve, lumbar spinal cords were dissected and fixed by immersion overnight at 4°C, in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (Renno et al., 2017; Hussain et al., 2018). Every 15th section of the spinal cord was stained for Cresyl Violet staining and immunostained for neurons using Primary antibodies for NeuN, GFAP and GAP 43. 5 µm thick paraffin sections of the spinal cord, were cut and mounted on Poly-L-lysine coated slides and kept overnight for drying. The tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol and then taken to the water. Endogenous peroxidase activity was quenched by treating sections with 3% hydrogen peroxide for 15 min, followed by 30 min incubation in 50 mM glycine and 0.1% sodium borohydride and then washed with PBS (Ph 7.4). Non-specific binding of antibodies was blocked by treating the sections with 5% normal goat serum. Sections were incubated with Primary antibodies (Anti-NeuN, Clone A60, Mouse Monoclonal Antibody, Cat# MAB377, Millipore. Billerica, Massachusetts, United States), Anti-GFAP antibody (GA-5: sc-58766, Mouse Monoclonal – Santa Cruz Biotechnology) or Anti-GAP-43 Antibody (B-5: sc-17790 – Santa Cruz Biotechnology) overnight at 4°C. Sections were then washed with PBS and treated with biotinylated goat anti-mouse IgG [(1:200), Vector Labs, Burlingame, CA, United States)] and 1% normal goat serum for 2 h at room temperature. Slides were then washed three times with PBS and treated with Avidin-Biotin Complex (Vector Labs, PK-6200, Burlingame, CA, United States) along with 0.1% Tween 20 for 1 h at room temperature. Sections were color developed with 3-diaminobenzidine as a chromogen (DAB kit, SK-4100, Vector Labs, Burlingame, CA, United States), for 30 s or until the desired brown color was obtained as seen under the microscope. Then slides were washed with distilled water, counterstained in hematoxylin for 5 min, followed by bluing under tap water for 5 min. The slides were dehydrated in graded ethanol and cleared in xylene. Finally, a coverslip was mounted on top of sections using DPX mountant for histology. For Cresyl violet staining, sections were mounted on gelatin-coated slides, air-dried overnight. Sections were hydrated in graded ethanol and stained with 0.1% cresyl violet stain. Slides were dehydrated in graded ethanol and cleared in xylene and mounted with DPX mountant for histology (44581, Sigma) (Renno et al., 2017; Hussain et al., 2018).

The number of NeuN labeled neurons in the ventral and dorsal grey horns of the spinal cord were counted using Cell Sens Dimension software. From each rat, ten sections were selected for neuron quantification. The spinal cord region under analysis was focused at ×40 magnification, and an image was transferred to a computer monitor with a high-resolution digital Nikon camera attached to an Olympus microscope (DP-72). The total number of neurons in the entire ventral and dorsal grey horns were counted. Slides were coded to avoid observers’ bias. The mean number of neurons per section was calculated for statistical analysis (Renno et al., 2015; Renno et al., 2016; Hussain et al., 2018).

The GFAP and GAP-43 immunoreactive staining intensity was measured with Cell Sens Dimension software. From each rat, ten sections were randomly selected for intensity measurement. The 10 sampling results per animal were averaged then the averages of five animal data per group were statistically analyzed. The total intensity in the entire ventral and dorsal grey horns was measured, and the mean GFAP and GAP-43 immunoreactive intensity per section was calculated for statistical analysis (Renno et al., 2015; Renno et al., 2016; Hussain et al., 2018).