Mouse anti mkp 1

After being lysed and centrifugated, the supernatant was determined by BCA kit. After being normalized and boiled with loading buffer, the proteins were separated and transformed into PVDF membrane (Millipore, Burlington, Massachusetts). After being treated with 5% bovine serum albumin for 1 hour, membranes were incubated with primary antibodies in 1% bovine serum albumin. In this study, rabbit anti-p-p38MAPK, p38MAPK, p-ERK1/2, ERK1/2, RXRα (Cell Signaling Technology, Danvers, Massachusetts; 1:1,000), rabbit anti-p-JNK1/2 (Abcam, Cambridge, UK; 1:2,000), JNK1/2 (Abcam; 1:5,000), mouse anti-MKP-1 (Santa Cruz Technology, Santa Cruz, California; 1:500), goat anti-iba-1 antibody (Abcam; 1:1,000), rabbit GAPDH and anti-β-actin (ABclonal Technology, Woburn, Massachusetts; 1:5,000) were used. After being washed, membranes were treated with horseradish peroxidase-linked antirabbit or mouse secondary antibodies (Cell Signaling Technology, 1:1,000) for 1 hour. Signals were visualized by with horseradish peroxidase substrate (Millipore; Luminata Forte).

The ispilateral L4 to L5 spinal cords on day 7 were treated with 4% paraformaldehyde for 8 hours and 30% sucrose overnight at 4°C. Then, samples were embedded and sliced. After being washed and treated with .3% Triton X-100 for 20 minutes, they were incubated with 5% donkey serum in phosphate-buffered saline for 1 hour and primary antibody in 1% donkey serum overnight. Goat anti-iba-1 antibody (Abcam; 1:200) were used. After being washed again, sections were treated with donkey antigoat IgG Dylight594 (1:200; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania). Images were taken at 100× magnification (Leica DM500B, Wetzlar, Germany). To detect colocalization of RXRα/MKP-1 and RXRα/Iba-1, rabbit anti-RXRα (1:200, Cell Signaling Technology), Goat anti-iba-1 antibody (Abcam; 1:200) and mouse anti-MKP-1 (1:50, Santa Cruz Technology) were used as primary antibodies in 1% donkey serum. Secondary antibodies were donkey antirabbit IgG Dylight488 (1:200; Jackson ImmunoResearch Laboratories), donkey antigoat IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories), and donkey antimouse IgG Dylight594 (1:200; Jackson ImmunoResearch Laboratories).