Tissues were embedded in paraffin and sectioned, and then dewaxed with xylene and hydrated with gradient ethanol solution. H2O2 methanol solution (3%) was added for inactivation treatment for 20 min, high temperature antigen in citrate buffer solution (pH 6.0) was used for thermal repair for 10 min, and 5% BSA was used for blocking treatment for 20 min. Rabbit anti rat Nogo-A (1:200, orb337377, Biorbyt, UK) and NgR (1:500, orb185998, Biorbyt) polyclonal antibodies were added and reacted overnight at 4°C. After rewarming, goat anti-rabbit IgG labeled with horseradish peroxidase (1:1000, orb21465, Biorbyt, UK) was incubated with the secondary antibody. After DAB staining, slides were dyed again, then dehydrated, cleared, and finally sealed. The slides were observed under the optical microscope (400×, Olympus). Analysis was done with ImageJ software (NIH, USA), and the results are reported as the percentage of positive cells (%).

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