Immunohistochemistry was performed as previously described (36, 38, 63). In brief, animals were deeply anesthetized with isoflurane and subsequently perfused transcardially with phosphate-buffered saline (PBS), followed by a fixative containing 4% paraformaldehyde. Tissue samples were sectioned at a thickness of 15 μm using a Leica CM1950 cryostat (Leica Microsystems Inc., Germany). Subsequently, tissue sections were washed in PBS and blocked for 1 hour in 0.25% Triton X-100 and 5% normal goat serum. After overnight incubation at 4°C with primary antibodies including rabbit anti-TrkB (1:400 dilution, Abcam; catalog no. ab18987), mouse anti-CGRP (1:500 dilution, Abcam; catalog no. ab81887), or mouse anti-NF200 (1:600 dilution, Sigma-Aldrich; catalog no. N2912), the sections were visualized with IB4-fluorescein isothiocyanate (5 μg/ml; Sigma-Aldrich; catalog no. L2895), goat anti-rabbit IgG conjugated to Cy3 (1:300 dilution, Abcam; catalog no. ab97075), or goat anti-mouse IgG conjugated to fluorescein isothiocyanate (1:300 dilution, Abcam; catalog no. ab6785) for 2 hours at room temperature. The slices were examined under an upright fluorescence microscope (104C, Nikon), and images were captured with a CoolSNAP HQ2 charge-coupled device camera (Photometrics). Negative controls were only incubated with the secondary antibody and did not show any specific immunostaining.

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