Immunohistochemical Staining of Spinal Cord and Nerve Tissue

For immunohistochemical staining, spinal cord or nerve tissue was prepared into 12 µm slices. The slices were fixed with 2% PFA in PBS (pH 7.4) for 20 min. After washing the slices for 5 min with PBS, they were permeabilized in PBST (PBS + 0.1% triton X) for 10 min. Then, samples were blocked for 45 min in 3% BSA in PBST. As first antibodies, GFAP (NB300-141, novusbio, Centennial, CO, USA) in 1:1000 dilution for spinal cord or F4/80 (ab6640, abcam, Cambridge, UK) and CD11b (ab133357, abcam) in 1:100 dilution for nerve were used. After incubation over night at 4 °C, the second antibodies, anti-rabbit Cy 3 (C2306-1ML, Sigma, Deisenhofen, Germany) and anti-rat AF488 (ab150157, abcam), were incubated in a dilution of 1:1000 at room temperature for 1 h. Afterwards, the slices were stained with DAPI 1:1000 (6335.1, Carl Roth, Karlsruhe, Germany). Pictures of the stained slices were taken with the fluorescence microscope Observer.Z1 (Carl Zeiss, Oberkochen, Germany) [31 (link)].