Journal: Journal of Histochemistry and Cytochemistry

Article Title: Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ

doi: 10.1369/0022155418756848

Figure Lengend Snippet: Detection of vRNA and vDNA by ISH using RNAscope. ACH2 cells (A and B) and Thymus/Liver implants from NSG-BLT mice 9 weeks after intraperitoneal inoculation with HIV-1 JR-CSF (C and D) were tested with the RNAscope 2.5 HD Duplex Reagent Kit, using probes for vRNA and vDNA (A and C), or scrambled sequence probes for vRNA and vDNA (B and D), vRNA (black), vDNA (red), and nuclei (blue). A cytospin of ACH2 cells are stained for vDNA (E). The red arrow in A shows a vDNA+/vRNA+ cell. Red circles in C highlight vDNA. vDNA background was seen in B and D (shown by red circles). The blue arrow in E shows cells with multiple vDNA signals. Main images are at 12× magnification, and inset images at 40×. Scale bar for inset image = 20 µm. Abbreviations: vRNA, HIV-RNA; vDNA, HIV-DNA; ISH, in situ hybridization; NOD, nonobese diabetic; SCID, severe combined immunodeficiency; NSG, NOD SCID gamma; BLT, bone-marrow/liver/thymus.

Article Snippet: The use of other antibodies will require optimization. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Primary antibodies: anti-CD3 [(rabbit) (UCHT1) (Dako) (1:100)]; anti-CD68 [(mouse) (NCL-CD68-KP) (Leica; Buffalo Grove, IL) (1:100)]; anti-CD163 [(mouse) (NCL-CD163) (Leica) (1:100)]; anti-CD21 [(mouse) (2G9) (Thermo Fisher) (1:10)]; anti-p24 [(mouse) (Kal-1, also known as H-11) (Dako) (1:50)] Secondary antibodies: anti-mouse [(DyLight 649) (DL-2649) (Vector) (1:250)]; anti-rabbit [(Alexa Fluor 594) ( {"type":"entrez-nucleotide","attrs":{"text":"A21207","term_id":"583479","term_text":"A21207"}} A21207 ) (Life Tech) (1:250)]; Polink-1 polymer horseradish peroxidase (HRP) anti-mouse IgG (GBI Labs; Mukilteo, WA) Advanced Cell Diagnostics (ACD; Newark, CA) RNAscope Probes list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HIV-RNA and HIV-DNA Singleplex assays: V-HIV1-Clade B sense-78 probe pairs (425531); V-HIV-Clade B antisense-78 probe pairs (416111) HIV-RNA and HIV-DNA duplex assays: HIV-non-gagpol antisense-C1-40 probe pairs (317711); HIV-gagpol-sense-C2-40 probe pairs (317701) 2-Plex positive control (320741) ACD RNAscope Kits list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 RNAscope 2.5 HD Singleplex Red or Brown Kit RNAscope 2.5 HD Duplex Reagent Kit RNAscope 2.5 HD Custom-Duplex Reagent Kit with dual HRP endpoints RNAscope Multiplex Fluorescent Kit, version 2 [requires TSA (Perkin Elmer) and HRP blocker (ACD)] Buffers and Solutions list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 TBS 1×: Tris-HCl 1.5M, Tris-Base 0.4M, NaCl 15 M dissolved in distilled Milli-Q water (MilliporeSigma; Billerica, MA) (pH 7.7).

Techniques: RNAscope, Sequencing, Staining, In Situ Hybridization

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ

doi: 10.1369/0022155418756848

Figure Lengend Snippet: Overcoming the optical barrier of chromogenic ISH. A single FFPE section from an HIV+ human rectal biopsy was stained for vRNA-ISH using the RNAscope 2.5 Brown kit, followed by TSA-Cy3 amplification of HRP-labeled vRNA and multiplexed IF for CD3 and CD68/CD163 (all fluorescent mIFISH)—DAPI (IF, blue, A); vRNA (TSA-Cy3, white, B); CD3 (IF, green, C); and overlay (D; all fluorescent mIFISH; 1 slide, B–D). The red circles in the inset images highlight a single CD3+ cell shown to be highly positive for vRNA. Scale bar = 50 µm. Abbreviations: ISH, in situ hybridization; FFPE, formalin-fixed paraffin-embedded; vRNA, HIV-RNA; TSA, tyramide signal amplification; HRP, horseradish peroxidase; IF, immunofluorescence; mIFISH, multiplexed IF and ISH; DAPI, 4′,6-diamindino-2-phenylindole.

Article Snippet: The use of other antibodies will require optimization. list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Primary antibodies: anti-CD3 [(rabbit) (UCHT1) (Dako) (1:100)]; anti-CD68 [(mouse) (NCL-CD68-KP) (Leica; Buffalo Grove, IL) (1:100)]; anti-CD163 [(mouse) (NCL-CD163) (Leica) (1:100)]; anti-CD21 [(mouse) (2G9) (Thermo Fisher) (1:10)]; anti-p24 [(mouse) (Kal-1, also known as H-11) (Dako) (1:50)] Secondary antibodies: anti-mouse [(DyLight 649) (DL-2649) (Vector) (1:250)]; anti-rabbit [(Alexa Fluor 594) ( {"type":"entrez-nucleotide","attrs":{"text":"A21207","term_id":"583479","term_text":"A21207"}} A21207 ) (Life Tech) (1:250)]; Polink-1 polymer horseradish peroxidase (HRP) anti-mouse IgG (GBI Labs; Mukilteo, WA) Advanced Cell Diagnostics (ACD; Newark, CA) RNAscope Probes list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 HIV-RNA and HIV-DNA Singleplex assays: V-HIV1-Clade B sense-78 probe pairs (425531); V-HIV-Clade B antisense-78 probe pairs (416111) HIV-RNA and HIV-DNA duplex assays: HIV-non-gagpol antisense-C1-40 probe pairs (317711); HIV-gagpol-sense-C2-40 probe pairs (317701) 2-Plex positive control (320741) ACD RNAscope Kits list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 RNAscope 2.5 HD Singleplex Red or Brown Kit RNAscope 2.5 HD Duplex Reagent Kit RNAscope 2.5 HD Custom-Duplex Reagent Kit with dual HRP endpoints RNAscope Multiplex Fluorescent Kit, version 2 [requires TSA (Perkin Elmer) and HRP blocker (ACD)] Buffers and Solutions list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 TBS 1×: Tris-HCl 1.5M, Tris-Base 0.4M, NaCl 15 M dissolved in distilled Milli-Q water (MilliporeSigma; Billerica, MA) (pH 7.7).

Techniques: Staining, RNAscope, Amplification, Labeling, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Immunofluorescence