Danielsohn P, Nolte A
Histochemistry. 1987;86(3):281-5. doi: 10.1007/BF00490259.
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.
通过使用溴代双胺作为硫醇基团的荧光标记剂，可以在无脊椎动物神经组织的半薄切片中，尤其是腹足纲动物的半薄切片中，显示含硫神经肽。半薄切片显示出标记肽的明亮荧光，并且如果需要出色的分辨率，则应使用半薄切片。在我们的实验条件下，三种溴代双胺（MB：单溴代双胺，DB：二溴代双胺，MQ：单溴三甲基铵双胺）均给出了阳性结果。选择二溴代双胺（DB）是因为其应用更方便。在腹足纲动物中，与经典的神经分泌染色方法相比，溴代双胺技术似乎是最特异且最灵敏的方法。含硫氨基酸的神经肽可以很容易地在神经细胞体、轴突和轴突膨体中显示出来。我们推测存在一些用经典方法无法染色的神经元，而通过溴代双胺技术可以将它们鉴定为肽能神经元。观察到荧光强度略有降低（褪色）。因此，测定了二溴代双胺反应产物的褪色速率；使用Neofluar 10/0.30物镜时，仅在1小时后荧光强度才降低50%。然而，我们认为，如果考虑特殊参数，对标记的神经肽进行比较定量应该是可行的。
