In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes.
We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.
Jirikowski
G F
GF
Department of Neuropharmacology, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Ramalho-Ortigao
J F
JF
Kesse
K W
KW
Bloom
F E
FE
D000818:Animals; D001921:Brain / Q000378:metabolism*; D001973:Bromodeoxyuridine; D004853:Epoxy Resins; D005260:Female; D007150:Immunohistochemistry; D008867:Microtomy; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000235:genetics*
D004853:Epoxy Resins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014667:Vasopressins; C004875:EPON; D001973:Bromodeoxyuridine