Fluorescent in situ hybridization technique for cell type identification and characterization in the central nervous system.
Central nervous system consists of a myriad of cell types. In particular, many subtypes of neuronal cells, which are interconnected with each other, form the basis of functional circuits. With the advent of genomic era, there have been systematic efforts to map gene expression profiles by in situ hybridization (ISH) and enhancer-trapping strategy. To make full use of such information, it is important to correlate "cell types" to gene expression. Toward this end, we have developed highly sensitive method of fluorescent dual-probe ISH, which is essential to distinguish two cell types expressing distinct marker genes. Importantly, we were able to combine ISH with retrograde tracing and antibody staining including BrdU staining that enables birthdating. These techniques should prove useful in identifying and characterizing the cell types of the neural tissues. In this article, we describe the methodology of these techniques, taking examples from our analyses of the mammalian cerebral cortex.
Watakabe
Akiya
A
Division of Brain Biology, National Institute for Basic Biology, 38 Nishigonaka Myodaiji, Okazaki, Japan. watakabe@nibb.ac.jp
Komatsu
Yusuke
Y
Ohsawa
Sonoko
S
Yamamori
Tetsuo
T
D000818:Animals; D002490:Central Nervous System / Q000166:cytology*; D002540:Cerebral Cortex / Q000166:cytology; D004076:Digoxigenin; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D005968:Glutamate Decarboxylase / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000737:chemistry; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D050598:Vesicular Glutamate Transport Protein 1 / Q000235:genetics
D005456:Fluorescent Dyes; D016372:RNA, Antisense; D012333:RNA, Messenger; C493465:Slc17a7 protein, mouse; D050598:Vesicular Glutamate Transport Protein 1; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin