Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the light- and electron-microscopic levels.
With the aim of localizing proenkephalin mRNAs in neurons of the hypothalamic magnocellular dorsal nucleus of the guinea pig, we compared the in situ hybridization signals obtained on Vibratome sections with a method employing either a biotinylated or a digoxigenin-labeled oligonucleotide detected by means of the alkaline phosphatase reaction. Since the hybridization approach using the biotinylated probe was more sensitive than the digoxigenin method, the ultrastructural localization of hybrids in neurons of the magnocellular dorsal nucleus was studied by the use of the former procedure, and was further compared with results of in situ hybridization using a 35S-labeled probe. Biotin was detected via an amplified avidin-biotin-peroxidase complex. Radioactive hybrids were localized over extended cytoplasmic compartments rich in rough endoplasmic reticulum and also in nuclear indentations. The method based on biotinylated probe proved to be sensitive and provided high-resolution labeling in well-preserved specimens. Proenkephalin mRNAs were clearly localized within circumscribed cytoplasmic compartments. The immunoprecipitates were mainly observed within the rough endoplasmic reticulum, especially at the periphery of the cell. The reticulum was dominated by elongated parallel cisternae. The labeling also appeared in a paranuclear position, mainly in nuclear indentations. The labeling was found on the outer surface of the endoplasmic lamellae. The remainder of the reticulum was unlabeled. Neuronal processes were free of labeling.
Mitchell
V
V
INSERM, Unité 156, Lille, France.
Gambiez
A
A
Beauvillain
J C
JC
D000469:Alkaline Phosphatase; D000818:Animals; D001710:Biotin; D003593:Cytoplasm / Q000737:chemistry; D004076:Digoxigenin; D004721:Endoplasmic Reticulum / Q000737:chemistry; D004745:Enkephalins / Q000235:genetics*; D006168:Guinea Pigs; D007031:Hypothalamus / Q000737:chemistry*; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000737:chemistry; D015345:Oligonucleotide Probes; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D013462:Sulfur Radioisotopes
D004745:Enkephalins; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C029992:proenkephalin; D001710:Biotin; D000469:Alkaline Phosphatase; D004076:Digoxigenin