2.5. Measuring CGRP Expression in the Dorsal Root Ganglion by Immunohistochemical Examinations

After the last measurement of the pain behaviors, the rats were anaesthetized with pentobarbital injection (100 mg/kg) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS). The dorsal root ganglion (DRG) was dissected out and preserved in PBS for a further period of 3 days. Cryostat sections (20 μ thick) were cut in the transverse plane at −20°C and collected as free-floating sections in 0.1 M phosphate buffer saline (PBS). An average of 11-12 sections per rat was collected. Later, the sections were processed for immunohistochemical localization of CGRP. Following this, sections were directly exposed to primary antibody against CGRP (1 : 400; Calbiochem, USA) for 48 h. CGRP expression was visualized by the ABC method (Vector Laboratories, USA) using 3,3-diaminobenzidine (DAB) tetrachloride (DAB) as the chromogen. Finally, the sections were taken on slides and dehydrated followed by clearing.

Images of the stained sections were captured under the microscope and the images were saved in ProgRes Capture Pro 2.7 Image analysis software (Jenoptik, Germany). The area of the CGRP expression in these sections, which corresponded to the superficial part of the DRG, was selected by Image Pro Plus software to calculate the integral optical density (IOD).