Our multiplex immunofluorescent cell-based detection of DNA, RNA, and protein approach enables us to simultaneously visualize proteins and nucleic acids of different types and strandedness within single cells. Before seeding, incubate cover slips in ethanol for five minutes, followed by two two minute washes in PBS. After the second wash, completely submerged the cover slip in 20 micrograms per milliliter of poly-D-lysine for 30 minutes at room temperature.
At the end of the incubation, remove the excess matrix solution with two washes in PBS and dilute the cells of interest at 1 x 10 to the sixth cells per 50 microliters of PBS per cover slip concentration. Then seed 50 microliters of the cells of interest onto each poly-D-lysine coated cover slip for a 30 minute incubation at room temperature. At the end of the incubation, wash the cells three times in PBS before fixing them in 4%paraformaldehyde for 30 minutes.
At the end of the incubation, wash the cells two times in PBS before permeabilizing the cells with 500 microliters of 0.1%Tween 20 in PBS for 10 minutes at room temperature. At the end of the incubation, wash each cover slip two times with 500 microliters of fresh PBS per wash. To immobilize the cover slip onto a glass slide, place a small drop of clear nail polish onto a sterilized glass slide and place one edge of the cell-free side of the cover slip onto the drop of nail polish and lower the cover slip onto the slide cell side facing up.
Add a few drops of PBS onto the immobilized cover slip to prevent the cells from dehydrating and use a hydrophobic barrier pen to draw a circle around the outside of the cover slip. When the barrier has been drawn, replace the PBS with 100 microliters of protease III and place the slide into a 40 degree Celsius incubator for 15 minutes. At the end of the incubation, wash the slides two times in fresh PBS for two minutes per wash with rocking.
After the second wash, dilute the probe of interest in 200 microliters of hybridization buffer at 67 degrees Celsius for 10 minutes. At the end of the incubation, add 50 microliters of the probe solution to each cover slip and place the slides into a humidified 40 degree Celsius oven for two hours. At the end of the incubation, wash the slides two times with fresh wash buffer and rocking for two minutes per wash.
After the second wash, tap the slides to remove the excess buffer and sequentially treat the cover slips with one drop of the amplifiers for fluorescent channels 1, 2, and 3 for a 30 minute, 40 degree Celsius incubation per amplifier with two two minute washes in fresh wash buffer with rocking per wash between treatments. After the last wash, add a drop of the fluorescent channel 4 amplifier to the cover slip for a 15 minute incubation in the 40 degree Celsius oven and wash the slide two times with wash buffer and one time with PBS as demonstrated. For immunostaining of the proteins of interest, after the last wash, treat each cover slip with 200 microliters of blocking buffer for a one hour incubation at room temperature.
At the end of the incubation, decant the blocking buffer and add 200 microliters of the primary antibody of interest diluted in PBS plus Tween 20 supplemented with 1%bovine serum albumin. After a one hour incubation at room temperature, wash the slides with two 10 minute washes in fresh PBS plus Tween 20 with shaking per wash before labeling the samples with an appropriate secondary antibody for one hour at room temperature. At the end of the incubation, wash the slides two times with fresh PBS plus Tween 20 for 10 minutes with shaking per wash.
After the second wash, counter stain the nuclei with an appropriate nuclear dye for one minute at room temperature, followed by two washes in PBS for 10 minutes at room temperature with shaking. To mount the samples for imaging, place one drop of antifade solution onto one new sterile glass slide per sample and spread the solution to cover an area approximately the size of the cover slips. Detach the cover slips from their slides and submerge the cover slips in PBS.
Use forceps to remove any residual nail polish and dry the back of the cover slip with a lab wipe. Then gently place each cover slip sample side down onto the antifade solution and allow the samples to dry overnight. As a proof of concept of this procedure, HIV-1 DNA, RNA, and protein were simultaneously labeled and visualized microscopically at the single cell level.
In this example, two HIV-1, DNA genomes can be visualized within a single cell as they are actively transcribing the viral RNA. The viral RNA was exported through the nuclear pore complex and the viral protein was synthesized in the cytoplasm. As observed, very little to no cross-reactivity is observed between the probe sets across the two viruses, despite labeling with two retroviruses with a potential for probe cross-reactivity.
In addition, viral RNA staining can be eliminated if the cells are treated with RNase during the sample preparation. The mean integrated fluorescence intensity of the antibody signal per cell in each image can then be quantified. As observed, the amount of HBV pre-genomic RNA and total HBV RNA increases over the course of the HBV infection.
HCV infection imaging can also be performed via confocal microscopy using a 60X oil immersion objective to detect the fluorophores of interest. This highly specific approach also allows the tracking on viral or cellular processes with a high spacial temporal resolution. In addition, this technique can be used for study multiple viruses and other pathogens.
