NPY- and C-PON-immunoreactive substances in the central nervous system of Helix pomatia.

The distribution of neuropeptide-tyrosin (NPY)- and C-flanking peptide of neuropeptide-tyrosine (C-PON)-immunoreactivities in the central nervous system of the pulmonate gastropod, Helix pomatia, was investigated. NPY- and C-PON-like substances were localized in neuronal somata and neuntes, but were not co-localized within the same cells. NPY-immunoreactive substances were also found in endocrine/paracrine like cells located in the epineurium. C-PON and NPY, both reduced serotonin activated isometric contractions of Helix aorta, suggesting that they may act as modulators in the control of the vascular system.

Application of silver acetate autometallography and gold-silver staining methods for in situ DNA hybridization.

In situ hybridization using biotinylated DNA probes has become an important tool in histopathology. It is well known that the sensitivity of the methods used to demonstrate viral DNA in formalin-fixed and paraffin-embedded specimen depends strongly on the detection system used. In the present study, an optimized in situ DNA hybridization protocol was combined with four different approaches of gold-silver staining methods. For silver enhancement, the recently described method of silver acetate autometallography, a technique allowing highly efficient development without the necessity of dark room illumination has been used. The most efficient detection method found in our experiments was the use of gold-adsorbed anti-biotin antibodies with subsequent silver enhancement. This staining procedure can be completed in 5 hours including hybridization and is a highly sensitive alternative to peroxidase and alkaline phosphatase detection systems.

Argyrophilic nucleolar organizer regions. A revised version of the Ag-NOR-staining technique.

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.