TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries.

Abstract

Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in aspecific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS celltype in the mixed culture and animal models of the CNS diseases and injuries.