Immunohistochemical Detection of proNGF

Sections (4 microns) were prepared from archival, formalin-fixed, paraffin-embedded tissue blocks immediately prior to staining. Immunohistochemistry was performed using the Ventana Discovery automated slide stainer (Roche Medical Systems, Tucson, AZ, USA). The primary antibody was an anti-rabbit polycloncal proNGF antibody (Ab9040, Merck Millipore, Darmstadt, Germany), which has been previously validated in our laboratory [11 (link)] and was reoptimised for automated tissue processing at a dilution of 1/350. Antigen-retrieval was performed using Ribo-CC solution, (pH 6, Ventana, Roche, North Ryde, NSW, Australia), with primary antibody incubation for 32 min, secondary anti-rabbit HQ for 16 min at 37 °C (Ventana, Roche, North Ryde, NSW, Australia) and tertiary anti-HQ (Ventana, Roche, North Ryde, NSW, Australia) for 16 min at 37 °C. Manual counterstaining was performed using Mayer’s haematoxylin for 10 s and Scott’s Tap Water for 30 s, followed by dehydration, clearing and mounting. Negative controls were prepared using nonspecific IgG isotype controls, and without the addition of primary antibody (Appendix A).