AN IMPROVED PROCEDURE FOR THE IMMUNOHISTOCHEMICAL LOCALIZATION OF NERVE GROWTH FACTOR-LIKE IMMUNOREACTIVITY

Nerve growth factor (NGF) is a survival factor required by a number of neuronal populations including most post-ganglionic sympathetic neuro nes. NGF has been detected and quantified in many tissues but there is little information regarding its cellular localization. Although it h as been argued that histological detection has proven difficult due to the low levels of NGF present, other factors may contribute to preven t its identification. In the present study, we report a method for the histological detection of NGF-like immunoreactvity in the rat superio r cervical ganglia (SCG). Adult Wistar-Kyoto rats were perfused briefl y with either a high or low pH buffer prior to fixation and routine im munohistochemistry. Polyclonal antibodies to native mouse NGF used in the present study recognized mouse NGF but not recombinant human neuro trophin 3 (rhNT3) or brain-derived neurotrophic factor (rhBDNF) by imm unoblot analysis. NGF-like immunoreactivity was localized to most symp athetic neurones. Immunoreactivity was detected in the cytoplasm with dense labelling around nuclei. No stain was seen in sections incubated with normal sheep IgG or from animals perfused with phosphate buffer (pH 7.4) prior to fixation. In addition, axotomy resulted in the disap pearance of NGF immunoreactivity which was confirmed by biochemical qu antification. Finally, no NGF immunoreactivity was found in neurones o f rats treated systemically with NGF antiserum 3 days earlier. Possibl e mechanisms underlying the improvement of NGF immunohistochemistry by pH manipulation before fixation are discussed.