Neuronal nuclei (neun)

The sections were placed in boiling 0.01 M citric acid (pH = 6) for antigen retrieval for 15 minutes. The repaired sections were blocked with goat serum blocking solution at 4°C for 2 hours, and the following primary antibodies were added to incubate overnight at 4°C: anti-nuclear factor kappa-B (NF-κB) (1:1000, Abcam, Cambridge, UK) and anti-tumor necrosis factor-α (TNF-α) (1:1000, Abcam, Cambridge, UK). On the next day, a fluorescent secondary antibody Alexa Fluor 568 (1:400; Life Technology, USA) was added to the spinal cord tissue, and the tissues were combined for 2 hours at room temperature. Subsequently, the sections were incubated with a second primary antibody (anti-neuronal nuclei [NeuN], 1:400; Novus Biologicals) for 2 hours at room temperature and then the tissues were incubated at room temperature for 2 hours with fluorescent secondary antibody Alexa Fluor 488 (1:400; Life Technology, USA). Next, the nuclei were counterstained by staining with 4′,6-diamidino-2-phenylindole (1:1000) and sealed with a neutral gum (Sigma-Aldrich, St. Louis, Missouri, USA). Tissue staining was observed under a fluorescence microscope (Leica, Heidelberger, Germany). Neurons in the anterior horn of the spinal cord were observed to evaluate changes in the inflammatory response of neurons.