title: Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.
abstract: To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.
authors.affiliation: Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands.
authors.forename: R W
authors.initials: RW
authors.lastname: Dirks
authors.forename: A K
authors.initials: AK
authors.lastname: Raap
authors.forename: J
authors.initials: J
authors.lastname: Van Minnen
authors.forename: E
authors.initials: E
authors.lastname: Vreugdenhil
authors.forename: A B
authors.initials: AB
authors.lastname: Smit
authors.forename: M
authors.initials: M
authors.lastname: Van der Ploeg
mesh_terms: D015073:2-Acetylaminofluorene; D015100:3,3'-Diaminobenzidine; D000818:Animals; D015342:DNA Probes; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics; D007447:Invertebrate Hormones / Q000378:metabolism; D008195:Lymnaea / Q000032:analysis*; D009479:Neuropeptides / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010455:Peptides / Q000235:genetics; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000032:analysis*; D013462:Sulfur Radioisotopes
chemical_list: D015342:DNA Probes; D007328:Insulin; D007447:Invertebrate Hormones; D009479:Neuropeptides; D015345:Oligonucleotide Probes; D010455:Peptides; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C026880:egg-laying hormone, Mollusca; D015100:3,3'-Diaminobenzidine; D015073:2-Acetylaminofluorene