Immunohistochemical labeling for CGRP in DRG sections

Dorsal root ganglia (DRG) from segments L1-5 from both sides were excised separately, fixed at 4°C in 4% paraformaldehyde for 24 hours, embedded in paraffin, cut into 5 μm sections which were dewaxed and autoclaved for 15 min (120°C, 1 bar). For CGRP labeling we applied overnight at 4°C a mouse reactive anti-CGRP antibody (1:100; polyclonal against a synthetic rat Tyr-CGRP(23–37) conjugated to gamma globulin developed in goat; Cat.No BP022, Acris antibodies, Herford, Germany; RRID:AB_973533). Sections were incubated for 2 hours in biotinylated rabbit anti-goat antibody (1:200; DAKO, Glostrup, Denmark), then the avidin-biotin peroxidase complex (Vectastatin-Elite ABC Kit, Vector Laboratories, Burlingame, USA) was applied for 40 min. Sections were developed with Jenchrom px blue (JenLab, Jena, Germany). The immunohistochemical labeling was evaluated as previously described [16]. In each second section the proportion of neuronal profiles with CGRP-like immunoreactivity (IR) was determined using a light microscope coupled to an image analyzing system (AxioVision, Zeiss). For each mouse the average proportion of labeled neuronal profiles was calculated. Per mouse at least 100 neuronal profiles with a visible nucleus were counted. In a control experiment the primary antibodies were omitted.