The overall goal of combining nascent RNA-FISH with amino staining of viral latent proteins is to visualize active transcription coupled to viral transcription factory formation. This method can be used to help answer key questions in the fields of genetics, cell biology, virology, and cell biology virology. The main advantage of this technique is it utilizes an inducible viral mini-chromosome model to study the molecular details behind genomic architectural alterations.
Collect cells in a 1.5 milliliter micro-centrifuge tube. Centrifuge the cells for two minutes at 200 Volt gravity. Wash the cells three times with one milliliter of sterile DEPC PBS.
Next, re-suspend the cells in 1.2 milliliters of DEPC PBS. Place the cover slips into the bottoms of a six-well plate. Pipette 200 microliters of the cell DEPC PBS mixture onto each cover slip.
After the cells have settled for two minutes, aspirate excess DEPC PBS, leaving only a thin layer of cells. Carefully add one milliliter of fixing solution to each cover slip. Allow the cells to fix for 10 minutes.
Wash the cover slips three times with DEPC PBS. After washing, carefully add one milliliter of glycine DEPC PBS to a final concentration of glycine of 100 millimolar to each cover slip. Allow the cells to quench for five minutes.
Add 1.5 milliliters of DEPC PBS and place on a shaker for five minutes, and shake firmly but gently in hand for one minute. Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Carefully add one milliliter of permeabilizing solution to each cover slip.
Allow the cells to permeabilize for 15 minutes. Add 1.5 milliliters of DEPC PBS. Place the cells on a shaker for five minutes, and shake firmly but gently in hand for one minute.
Aspirate the excess DEPC PBS and be careful not to disrupt the cells. Label the microscope slides, and moisten a paper towel then line the bottom of a sealable plastic container with the towel. Next, prepare the primary antibody solution and multiply the recipe for each cover slip present.
Place 30 microliters of primary antibody solution onto each labeled microscope slide. Ensure the primary antibody concentration is correct by holding the corner of the cover slip and gently flicking it. Additionally, the corner of a tissue can be used to wick excess moisture.
Then place the cover slip onto the labeled microscope slides, being careful not to introduce bubbles. Cover the exterior of the container with plastic wrap. Move the container into an incubator set to 37 degrees Celsius for one hour.
During incubation, prepare the necessary reagents. After incubation, wash the cells three times with DEPC PBS in a six-well plate for five minutes each. Then, wash the cells with FISH wash buffer three times for five minutes each.
And keep the cover slip in FISH wash buffer until the secondary antibody, an intron probe mixture has been prepared. Next, clean the glass slides for primary antibody incubation for hybridization with soapy water. After they're dry, clean them with ethanol and a lab tissue.
Place 40 microliters of the secondary antibody and FISH intron probe with solution onto each glass slide. Place the cover slips with the cells onto the slides with the cells side facing down on top of the buffer. And be careful not to introduce bubbles.
Place the slides into a plastic container with a moist paper towel on the bottom. Wrap the plastic container in first plastic wrap, and then aluminum foil. And incubate the container with the slides.
After incubation, carefully slide the cover slip from the edge of the glass slide and place it back into six-well plates, facing up. Wash the cells with FISH wash buffer three times in a six-well plate for five minutes each. Next, wash the cells two times with two-fold SSC.
Add DAPI at one to 1000 concentration into the two-fold SSC and allow the solution to sit for five minutes at room temperature. Wash the cells twice with two-fold SSC. Clean the glass slides used for hybridization and add 10 microliters of mounting solution onto the glass slides.
Place the cover slips face down onto the mounting solution. With a laboratory tissue, gently remove the excess mounting solution, being careful not to squash the cells. Using nail polish, apply an ample layer around the edge of the cover slips and allow the polish to dry.
The sample is now ready for fluorescence microscopy. BCBL-1 cells were stained for LANA and K-Rta RNA to examine where actively transcribing viral episomes are and the heterogeneity of response to Kaposi's Sarcoma-associated Herpesvirus, or KSHV reactivation stimuli. The distinct K-Rta fluorescence in regions that closely match the distribution of viral episomes is evidence for active transcription taking place close to KSHV genomes.
In the same sample, cells can be observed which exhibit much weaker and diffuse K-Rta fluorescence that does not significantly overlap with LANA, illustrating the considerable variation and the degree of response to reactivation stimuli within a population of cells. Next, the effectiveness of thymidine synchronization was examined by visualizing the expression of viral protein which clearly demonstrated that synchronized cells responded more to the reactivation stimuli than the unsynchronized population. DAPI staining can serve several functions, this example clearly shows that there are three separate cells instead of one.
3D microscopy alongside this technique revealed ring-like RNA PII structures seen with KSHV genomes dotted on the periphery. With its development, the mini viral chromosome would provide unique tools for researchers to explore the special temporal organization of gene-expression and the regression of the cell nucleus.
