Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semi-thin sections.
We have designed a procedure to investigate vasopressin (AVP) gene expression on plastic-embedded tissue by using in situ hybridization to detect AVP mRNA and immunohistochemistry to detect AVP. Rat brain was fixed and vibratome slices were incubated with a 45-base synthetic oligonucleotide complementary to AVP mRNA labeled with 35S, embedded in Araldite, and cut into semi-thin serial sections that were either processed for autoradiography or treated with an AVP antiserum. The results show that AVP mRNA is detectable in magnocellular neurons of the supraoptic and paraventricular nuclei in both vibratome and semi-thin sections. Osmication after hybridization does not modify the signal. AVP mRNA is restricted to the cytoplasm of magnocellular neurons and to the proximal portion of certain processes. Neurons labeled with the AVP probe were also stained with the AVP antiserum. AVP mRNA quantity and the intensity of AVP immunoreactivity are not consistently related in neurons. At least two hypotheses must be considered to explain these differences: first, the procedure presently used could lead to a reaction intensity that does not exactly reflect the amount of antigen or mRNA present in cells; second, the difference observed may reflect the fact that transcriptional and translational events are not constantly linked and can be regulated differently from one AVP neuron to another. This method provides a way to detect mRNA on semi-thin sections together with antigenic molecules and to accurately investigate gene expression in complex tissues with optimal histological quality.
Guitteny
A F
AF
Laboratoire d'Histologie-Embryologie (Unité Associée au CNRS 040561), Faculté de Médecine, Besançon, France.
Böhlen
P
P
Bloch
B
B
D000818:Animals; D001127:Arginine Vasopressin / Q000032:analysis / Q000235:genetics*; D001923:Brain Chemistry; D003593:Cytoplasm / Q000032:analysis; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D008297:Male; D009474:Neurons / Q000032:analysis; D009693:Nucleic Acid Hybridization; D010286:Paraventricular Hypothalamic Nucleus / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013495:Supraoptic Nucleus / Q000032:analysis
D012333:RNA, Messenger; D001127:Arginine Vasopressin