In Situ Hybridization
a) Live cell imaging: Fluorescent dyes, such as SYTOX green (Molecular Probes), which is a high-affinity nucleic acid stain but cell-impermeable dye (Ex./Em = 500nm/523nm), will be used to examine cell viability during or after treatment. Such dyes will enter cells with compromised plasma membranes but will not those of live cells. Using a 40x or 60x water-immersion objective lens immediately after mixing the viruses and host cells, dual-excitation and dual-emission images will be collected simultaneously under a BioRad MRC-1024ES confocal laser scanning microscope equipped with Argon/Krypton lasers. A series of such images will be collected using a computerized time-course program (BioRad LaserSharp program). Quantitative analyses of images from at least 3 independent experiments of duplicate cell cultures will be performed to determine the effect of ???? on the rate of cell survival or apoptosis.
b) Immunochemical labeling: Cells growing in 8-well chambers exposed to different treatments will be fixed in 4% paraformaldehyde and will be used for immunochemical assays for apoptosis-related agents or proteins using standard single or double labeling immunofluorescence microscopy.