Journal: Journal of visualized experiments : JoVE

Article Title: Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

doi: 10.3791/61843

Figure Lengend Snippet: 4. Coverslip immobilization on glass slide and protease treatment to remove nucleic acid binding proteins from fixed viral/cellular nucleic acid to improve hybridization efficiency

Article Snippet: Let protease reach RT for 10 min before permeabilization. table ft1 table-wrap mode="anchored" t5 Materials Name Company Catalog Number Comments 4% PFA 50% dextran sulfate Amreso 198 For DNA hybridization buffer 50X wash buffer ACD Bio 320058 6-well plates Amplifier 1-FL ACD Bio Amplifier 2-FL ACD Bio Amplifier 3-FL ACD Bio Amplifier 4-FL ACD Bio Consult Amp-4 table in protocol Anti-HCV NS5a antibody Abcam ab13833 Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4 Anti-HIV-1 p24 monoclonal antibody NIH AIDS Reagent Program 3537 Anti-Mov10 antibody Abcam ab80613 Rabbit polyclonal Anti-PB1 antibody GeneTex GTX125923 Antibody against flu protein Bovine serum albumin Blocking reagent for immunostaining Cell media with supplements Media appropriate for cell model Coverslips DAPI ACD Bio Nuclear stain (RNAscope kit from ACD Bio) Dulbecco’s phosphate buffered saline (1X PBS) Gibco 14190250 No calcium and magnesium Ethylene carbonate Sigma E26258 Fetal bovine serum (FBS) Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS) Fisherbrand colorfrost plus microscope slides Fisher Scientific 12-550-17/18/19 Precleaned HCV-GT2a-sense-C2 probe ACD Bio 441371 HCV(+) sense RNA probe HIV-gagpol-C1 ACD Bio 317701 HIV-1 cDNA probe HIV-nongagpol-C3 ACD Bio 317711-C HIV-1 RNA probe HybEZ hybridization oven ACD Bio 321710/321720 ImmEdge hydrophobic barrier pen Vector Laboratories H-4000 Nail polish For immobolizing coverslip to slide prior to protease treatment Nuclease free water Ambion AM9937 Poly-d-lysine (PDL) Coat coverslips in 20 μg/mL of PDL for 30 minutes Probe diluent ACD Bio 300041 For diluting RNA C2 or C3 probes Prolong gold antifade Invitrogen P36930 Protease III ACD Bio 322337 RNAscope® Probe- V-Influenza-H1N1-H5N1-NP ACD Bio 436221 RNase A Qiagen Secondary antibodies Slides Sodium chloride For DNA hybridization buffer Sodium citrate, pH 6.2 For DNA hybridization buffer Tween-20 For DNA hybridization buffer and PBS-T V-HBV-GTD ACD Bio 441351 Total HBV RNA V-HBV-GTD-01-C2 ACD Bio 465531-C2 HBV pgRNA probe V-HCV-GT2a probe ACD Bio 441361 HCV(−) sense RNA probe V-HTLV-HBZ-sense-C3 ACD Bio 495071-C3 HTLV-1 (−) sense RNA probe targetting HBZ V-HTLV1-GAG-C2 ACD Bio 495051-C2 HTLV-1 DNA probe V-HTLV1-GAG-POL-sense ACD Bio 495061 HTLV-1 (+) sense RNA probe V-Influenza-H1N1-H5N1-NP ACD Bio 436221 IAV RNA probe V-ZIKA-pp-O2 ACD Bio 464531 Zika(+) sense RNA probe V-ZIKA-pp-O2-sense-C2 ACD Bio 478731-C2 Zika(−) sense RNA probe Open in a separate window 4.

Techniques: Binding Assay, Hybridization, DNA Hybridization, Blocking Assay, Immunostaining, Staining, RNAscope, Saline, Microscopy, Plasmid Preparation