Goat anti mouse igg h l alexa fluor 405

Primary cell cultures were fixed for 15 min at room temperature with 3% formaldehyde/PBS. The fixed cultures were washed and permeabilized with 0.1% Triton X‐100/PBS at room temperature for 10 min, incubated with primary antibodies diluted in PBS at room temperature for 1 hr, washed with PBS, and then incubated with regular secondary antibodies conjugated to Alexa 405 (Life Technologies) for 1 hr at room temperature. The following primary and secondary antibodies were used: Anti‐SOX9 antibody (Merck Millipore, AB5535), Anti‐SOX5 antibody (Abcam, ab94396), Anti‐SOX6 antibody (Abcam, ab30455), Anti‐p21 antibody (Cell Signaling Technology, 2946), Goat anti‐Rabbit IgG H + L Alexa Fluor 405 (Thermo Fisher, A‐31556), Goat anti‐mouse IgG H + L Alexa Fluor 405 (Abcam, ab175660).

Flow cytometry was used to analyze the proportion of Mets forming cells in the injured area. Steps were as follows: the spinal cord tissue was cut into pieces in 4°C PBS, centrifuged, digested, and recentrifuged. After that, 200 μL PBS was added and the tissue homogenate was resuspended in the filter. Then, 2 μL Rabbit anti-histone H3 (citrulline R2 + R8 + R17) polyclonal antibody (1 μg/mL; Abcam) and Mouse anti-CD68 monoclonal antibody (1 : 100; Abcam) were added to the resuspended cells. The mixture was incubated 30 min on ice in dark and then it was centrifuged and the supernatant was removed. Then, the precipitation was washed with 500 μL PBS for 3 times, and 200 μL PBS was added to resuspend. Afterward, add 2 μL secondary antibody of Donkey Anti-Rabbit IgG H&L (Alexa Fluor 647) (1 : 2000; Abcam), and Goat Anti-Mouse IgG H&L (Alexa Fluor 405) (1 : 2000; Abcam), respectively, and incubate on ice for 30 min without light. Repeat centrifuging and washing for 3 times. The resorted cells were put into a flow cytometer (MACSQuant^® to Analyzer 10; Miltenyi Biotec, German) for flow detection. In the scatter plot of flow cytometry, CD68+ and CitH3+ double-positive cells were considered to be Mets forming cells.