Glial fibrillary acidic protein (GFAP) is a marker for astrocytes that is commonly used to evaluate the content of glial scars at the site of injury (Zhang et al., 2015).
Six weeks after surgery, three rats were perfused and sampled as described above after electrophysiological examination. The spinal cord in the C6 segment was fixed with 4% paraformaldehyde, dehydrated with 30% sucrose, frozen, and transversally sliced into 30 μm-thick sections. One section from every four sections was selected for examination. Primary antibody goat anti-rat GFAP antibody (1:200; Sigma) was added and incubated at 4°C overnight. Secondary antibody rabbit anti-goat Fluor 488 (Abcam, Cambridge, UK) was added and incubated at room temperature for 1.5 hours. All sections were observed under a fluorescence microscope (Carl-Zeiss Axioplan 2 imaging E, Aalen, Germany). ImageJ analysis software 1.42v (National institute of Health, Bethesda, MD, USA) was used to calculate the GFAP-positive staining area and total tissue area of the damaged dorsal horn of the spinal cord in the immunofluorescent images. The ratio of GFAP-positive cells to the total number of cells (the ratio of the GFAP stained area to the total tissue area) was calculated.
Six weeks after surgery, three rats were perfused and sampled as described above after electrophysiological examination. The spinal cord in the C6 segment was fixed with 4% paraformaldehyde, dehydrated with 30% sucrose, frozen, and transversally sliced into 30 μm-thick sections. One section from every four sections was selected for examination. Primary antibody goat anti-rat GFAP antibody (1:200; Sigma) was added and incubated at 4°C overnight. Secondary antibody rabbit anti-goat Fluor 488 (Abcam, Cambridge, UK) was added and incubated at room temperature for 1.5 hours. All sections were observed under a fluorescence microscope (Carl-Zeiss Axioplan 2 imaging E, Aalen, Germany). ImageJ analysis software 1.42v (National institute of Health, Bethesda, MD, USA) was used to calculate the GFAP-positive staining area and total tissue area of the damaged dorsal horn of the spinal cord in the immunofluorescent images. The ratio of GFAP-positive cells to the total number of cells (the ratio of the GFAP stained area to the total tissue area) was calculated.