The protocol is helpful for gaining quantitative insight into the temporal regulation of viral gene products, within the context of host cells, especially in relation to herpes viruses. This technique can help address research questions related to both host and viral RNAs and proteins. Moreover, this technique is compatible with simultaneous biochemical assays.
To adhere the cells to eight chamber slides, apply 200 microliters of an inherent cell suspension to each chamber of a sterile, eight chambered slide and allow seed growth for 12-24 hours, at 37 degrees Celsius, and 5%carbon dioxide. At the end of the incubation, aspirate the supernatant and unattached cells, and immediately fix the cells with pre-chilled 4%formaldehyde in PBS on ice, for 30 minutes. At the end of the fixation, wash the cells with three, five minute washes in 200 microliters of 4 degree Celsius PBS per wash.
After the last wash, permeabilize the fixed cells with 200 microliters of pre-chilled 0.5%Triton-X in PBS per well, for 10 minutes on ice. At the end of the permeabilization, carefully remove the chambers without cracking the slide, and rinse the cells with pre-chilled PBS. Block the rinsed cells with pre-chilled 4%BSA in PBS for 30 minutes at four degrees Celsius, followed by incubation with appropriate polyclonal primary antibody of interest in 0.1%BSA in PBS for one hour, at four degrees Celsius.
At the end of the incubation, wash the cells three times with fresh PBS and incubate the cells with an appropriate secondary antibody conjugated with a fluorophoric compatible with the FISH detecting antibody for one hour, at four degrees Celsius. After three PBS washes as demonstrated, fix the samples again in 4%formaldehyde in PBS for 10-15 minutes before a second permeablization as demonstrated. Then cover the slide with aluminum foil to preserve the fluorescent signal and prevent photobleaching.
And wash the cells with 2x saline sodium citrate, before applying 45 microliters of hybridization solution. After one hour at 37 degrees Celsius in a humidified chamber, add distilled water to the antisense oligonucleotides of interest to bring the denaturation volume to 10 microliters. Denature the oligonucleotides at 95 degrees Celsius for five minutes, before adding 35 microliters of fresh hybridization solution per slide.
Then incubate the samples from 10-24 hours in the humidified chamber at 37 degrees Celsius, protected with foil. The next day, wash the cells with two, 10 minute. 2X saline sodium citrate washes at room temperature, followed by one wash in 1X saline sodium citrate for 10 minutes, at 25 degrees Celsius.
After the last wash, fix the cells with pre-chilled 4%formaldehyde in PBS, for 10-15 minutes on ice, followed by three washes with fresh PBS. Next, permeabilize the samples as demonstrated, followed by incubation with anti-dioxigenin FTC and pre-chilled 0.1%BSA in PBS, for one hour at four degrees Celsius. At the end of the incubation, wash the slides three times in fresh PBX, and fix the samples with pre-chilled 4%paraformaldehyde in PBS, for 10-15 minutes at four degrees Celsius.
After three washes in PBS, label the nuclei with 0.4 micrograms per milliliter of DAPI and pre-chilled 0.5%Triton-X in PBS for 15 minutes on ice, followed by three final washes in PBS. Mount slides with fluorescent beads, as experimentally relevant and in appropriate mounting medium. After removing excess mounting medium with sterile wipes, seal one coverslip to each slide with multiple coats of clear nail polish, and use a fluorescent microscope to confirm successful execution of the experiment.
Images may then be taken on a confocal microscope to collect images of the samples within an hour to a week of mounting, at a 630 times magnification. These representative FISH and immunofluorescence results are semiquantitative and offer insight into oligonucleotide localization, rather than into comparisons between the intensities of the different fluorescent stains, because these experiments did not include a fluorescent bead in the slide preparation. In these experiments, the cytoplasmic and nuclear areas and their ratios were different for latent and lytic KSHV infected cells.
As illustrated in this figure, area is controlled in the nucleocytoplasmic ratio. When KSHV DNA replication is inhibited in the lytic phase, the early lytic protein of KSHV oligonucleotide transcript shifts to a predominantly cytoplasmic localization. KSHV polyadenylated RNA, however, localizes to specific nuclear sites despite the inhibition of viral DNA replication.
When removing the chambers from the slides, take care that there's very little adhesive residue on the tool, and use ethanol to permeablize the cells and weaken the adhesive. Biochemical assays such as QPCR, Western blot, and high-throughput sequencing, can be performed in tandem to augment the quantitative data collected from the micrographs.
