Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Abstract

Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient. In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons--not stainable by the classical methods--which can be identified as peptidergic ones by the bromobimane technique. A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.