CGRP Immunohistochemistry in DRG

The DRG fixed in 4% buffered formaldehyde was embedded in paraffin. The tissue block was sectioned at a thickness of 4-μm, and mounted on poly lysine coated slides. Four serial slides were deparaffinized and rehydrated sequentially, followed by the Streptavidin–Biotin Complex immunohistochemistry procedure as follows. The serial slides were incubated with rabbit-anti-CGRP (Abcam Inc., Cambridge, MA-02139-1517, USA; 1:100 diluted in PBS), and PBS (the negative control), respectively, and, sequentially, for 24 h at 4°C. Then, they were treated with secondary antibodies (SA1022-anti-rabbit IgG Kit, Wuhan Boster Biological Technology Ltd., Wuhan, China). Avidin-biotin complex Staining (Wuhan Boster Biological Technology Ltd., Wuhan, China) was visualized with diaminobenzidine (DAB) for 1 min at room temperature. All sections were dehydrated in graded ethanol series, made transparent in xylene, coverslipped, and air-dried. The slides were visualized with the images of the stained areas under a light microscope (Nikon ECLIPSE 80I, Nikon Corporation, and Tokyo, Japan), and 3 fields of each slide were observed with a 20 × objective lens. The numbers of CGRP positive cells were counted with the Image-Pro plus 6.0 system (Media Cybernetics, Inc., Bethesda, MD, USA). The mean values calculated from three sections represented the CGRP positive cells per rat.