Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.
We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.
Mitchell
V
V
INSERM U156, Lille, France.
Beauvillain
J C
JC
Mazzuca
M
M
D000818:Animals; D001345:Autoradiography; D004744:Enkephalin, Methionine / Q000032:analysis*; D004745:Enkephalins / Q000032:analysis*; D006168:Guinea Pigs; D007031:Hypothalamus / Q000737:chemistry*; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D009693:Nucleic Acid Hybridization; D011498:Protein Precursors / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*
D004745:Enkephalins; D011498:Protein Precursors; D012333:RNA, Messenger; C029992:proenkephalin; D004744:Enkephalin, Methionine