Determining Viral RNA Distribution in Host Cells by Fluorescence In Situ Hybridization

Abstract

Source: Vallery, T. K. & Steitz, J. A. Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells. J. Vis. Exp. (2019)

This video demonstrates fluorescence in situ hybridization to visualize viral RNA in virus-infected cells. It outlines the steps involved in probe hybridization, signal amplification, and confocal imaging.

Protocol

1. Fixation, Hybridization, and Visualization of Viral RNAs

1. Remove media and excess cells. Throughout this protocol, use vacuum suction to remove solutions and gentle micropipetting to add solutions.
   NOTE: The strength of a vacuum can be reduced by placing a 200 µL micropipette tip over the glass Pasteur pipette. Replace the micropipette tip between wash steps to prevent contamination. Each wash step must be performed quickly because it is imperative that the cells never dry out.
2. Immediately, fix the cells with pre-chilled 4% formaldehyde/PBS (phosphate-buffered saline) on ice for 30 min. Wash the cells three times with 200 µL 1x PBS cooled to 4 °C and incubate for 5 min at room temperature or on ice.
   1. Permeabilize the fixed cells with 200 µL of pre-chilled 0.5% Triton-X/PBS for 10 min on ice or 750 µL of pre-chilled 70% ethanol at 4 °C for 1 h (min) to 7 d (max).
      NOTE: All washes throughout this protocol are performed in the same manner unless otherwise specified. 70% ethanol loosens the glue between the chambers and the slide, which eases later separation, and also provides a significant pause in the protocol. Nonetheless, use paraffin film around the chamber slide to reduce evaporation and check the level of the ethanol in each chamber about every 8 h. 70% ethanol also flattens the cells, making a crisper image, while Triton-X does not dehydrate the cells and change the dimensions of the cell.
3. Remove chambers carefully to prevent cracking the slide.
   NOTE: Use a fresh removal device or one with very little leftover adhesive provided by the manufacturer, and gently ease the chambers off to prevent the slide from cracking. Using 70% ethanol as the permeabilizing reagent for 4 h greatly reduces the likelihood of cracking. In the case of a crack, continue the protocol on chambers not affected by the crack and be mindful of the higher oxidation rate of imperfectly sealed slides (i.e., decreased storage life).
4. Wash the cells with 2x SSC (saline sodium citrate) once and then apply 45 µL of hybridization solution consisting of 50% formamide, 10% dextran sulfate, 2x SSC, 0.1% bovine serum albumin (BSA), 500 µg/mL salmon sperm deoxyribonucleic acid (DNA), 125 µg/mL E. coli transfer RNA (tRNA), and 1 mM vanadyl ribonucleoside complexes. Incubate for 1 h at 37 °C in a humidity chamber that can be a 150 mm Petri dish with moistened sterile wipes.
   NOTE: Prepare fresh hybridization solution at least an hour before use. Dissolve the dextran sulfate in water first, vortexing frequently and incubating in a 37 °C water bath.
5. Calculate the amount of oligonucleotides required to achieve the suggested concentration of 25 µM in 35 µL of hybridization solution per chamber. Adjust the concentration of anti-sense oligonucleotide as needed. Add distilled water to the oligonucleotides to bring the denaturation volume to 10 µL.
   NOTE: Following the labeling reaction, the oligonucleotides are stored in the quenched solution containing 0.18 M potassium cacodylate, 23 mM Tris-HCl, 0.23 mg/mL BSA, 4.5 mM CoCl₂, 18 mM ethylenediaminetetraacetic acid (EDTA), 2.7 mM potassium phosphate, and 6.8 mM KCl, 45 µM 2-Mercaptoethanol, 0.02% Triton X-100, and 2% glycerol. The concentrations are high enough that dilution with water will bring the denaturation solution to concentrations near 1x TE (10 mM Tris-HCl and 1 mM EDTA), a standard oligonucleotide denaturation buffer.
6. Denature the digoxigenin (DIG)- and/or Alexa Fluor 594-labeled oligonucleotides at 95 °C for 5 min. Then add 35 µL fresh hybridization solution per intended chamber to the denatured oligonucleotides. If performing double fluorescence in situ hybridization (FISH), both sets of anti-sense oligonucleotides may be denatured and hybridized together.
7. Remove the pre-hybridization solution and then add the hybridization solution containing the labeled oligonucleotides to the cells. Incubate overnight in the humidity chamber at 37 °C with tin foil to protect the fluorophore-labeled oligonucleotides.
   NOTE: Incubation should be at least 10 h and not more than 24 h.
8. The next day, wash the cells twice with 2x SSC for 10 min at 37 °C and then twice with 1x SSC for 10 min at 25 °C.
9. Fix the cells with pre-chilled 4% formaldehyde/1x PBS for 10-15 min on ice. Then wash the cells with PBS three times and permeabilize for 1 h with pre-chilled 70% ethanol or for 10 min with pre-chilled 0.5% Triton-X/1x PBS at 4 °C.
10. Incubate the cells with 1:200 anti-digoxigenin (DIG)–fluorescein isothiocyanate (FITC) in pre-chilled 0.1% BSA/1x PBS for 1 h at 4 °C. Remove the antibody solution and wash three times with 1x PBS.
11. Fix with pre-chilled 4% formaldehyde/1x PBS for 10-15 min at 4 °C and then wash three times with 1x PBS.
12. Incubate the cells with 0.4 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) in pre-chilled 0.5% Triton-X/1x PBS for 15 min on ice and then wash three times with 1x PBS.
13. Mount slides with fluorescent beads (optional) and a mounting medium. Then seal the coverslip to the slide with clear nail polish.
14. Using a confocal microscope, collect images of the samples within an hour to a week of performing the protocol at 630x magnification. Apply multiple coats of nail polish to seal the cover slip and to prolong fluorophore life by reducing the rate of oxidation.
    NOTE: Do not use a DAPI-containing mounting medium. When collecting the images, include the scale bar on each image for later quantification. Fluorescent beads serve as controls of fluorescence intensity between slides and sample preparations. Acquire images at the midsection of the cell for two-dimensional (2D) quantification.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-DIG FITC	Jackson Lab Immunologicals	200-092-156
Dextran sulfate	Sigma Aldrich	D8906	Molecular biology grade
DIG-oligonucleotide tailing kit	Sigma Roche	#03353583910	2nd Gen
Eight-chamber slides	Nunc Lab Tek II	#154453	Blue seal promotes surface tension but separation by clear gel is also available.
Formamide	Sigma Aldrich	F9037	Molecular biology grade
Vanadyl ribonucleoside complexes	NEB	S1402S