A method for detection of a cytokine and its mRNA in the central nervous system of the developing rat.
We report here an effective and concise method to determine the localization of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, and its mRNA in the central nervous system of pre- and postnatal rats. This method allows for double staining to demonstrate localization of different molecules on the same tissue specimen at the levels of mRNA and proteins by in situ hybridization and immunohistochemistry, respectively. Additionally, the present method gives results more quickly than the conventional isotopic techniques. By use of this method, we carried out immunohistochemistry with an anti-rat MIF polyclonal antibody and demonstrated positive staining using the avidin-biotin complex method (ABC method). To detect its mRNA, we performed nonradioactive in situ hybridization using a digoxigenin (DIG)-labeled RNA probe prepared from a full length fragment of rat MIF cDNA. MIF was strongly expressed in the telencephalon on embryonic day 16. Non-radioactive in situ hybridization with a DIG-labeled RNA probe as well as the immunohistochemistry described here could be applicable to characterize localization of mRNA and proteins of different molecules on the same tissue specimen.
Suzuki
T
T
Central Research Institute, Hokkaido University School of Medicine, Sapporo, Japan.
Ogata
A
A
Tashiro
K
K
Nagashima
K
K
Tamura
M
M
Yasui
K
K
Nishihira
J
J
D000818:Animals; D001921:Brain / Q000196:embryology / Q000254:growth & development; D001923:Brain Chemistry / Q000235:genetics*; D018076:DNA, Complementary; D004076:Digoxigenin; D005260:Female; D005333:Fetus / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D015151:Immunoblotting; D007124:Immunoenzyme Techniques / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D008263:Macrophage Migration-Inhibitory Factors / Q000032:analysis* / Q000235:genetics*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344
D018076:DNA, Complementary; D008263:Macrophage Migration-Inhibitory Factors; D012333:RNA, Messenger; D004076:Digoxigenin