This technology enables the identification of signals that would be impossible to detect with wide-field fluorescence or standard confocal microscopy (for details of these improved technologies see (12,21C23))

This technology enables the identification of signals that would be impossible to detect with wide-field fluorescence or standard confocal microscopy (for details of these improved technologies see (12,21C23)). Example 1: As demonstrated in Figure 3, isolated CD4+ T lymphocytes from individuals with undetectable HIV replication as determined by ELISA (<15 pg/ml) and PCR (<20 copies/ml) were negative for HIV-p24 staining using regular immune staining (Figure 4, top panel). viremia to often to undetectable levels by antiretroviral therapy (ART), treatment is still not curative. A major obstacle to complete HIV eradication is the generation of viral reservoirs that sequester the virus in infected individuals (1C3). The best characterized HIV reservoir is a small population of resting CD4+ memory and na?ve T cells (1,2,4), but other reservoirs in macrophages and astrocytes (3C5) B-Raf-inhibitor 1 also have been described. Currently, identification and quantification of viral reservoirs is mainly performed by PCR or cell reactivation based technologies, but both detection systems have interpretation and technical difficulties, including the need for large amounts of blood, extensive time allocation, high cost, and significant differences in assay sensitivity (6C8). We have developed B-Raf-inhibitor 1 several comprehensive, integrated, and highly sensitive assays to analyze viral reservoirs by simultaneously examining integrated HIV DNA (sensitivity equal to one copy of HIV DNA per cell) or HIV mRNAs (sensitivity for few molecules) and viral proteins (sensitivity of few proteins, protocols described below). Because the detection is by imaging techniques, it does not require cell purification or amplification of the HIV components for the identification of a small number of viral reservoirs among millions of uninfected cells. We achieved this sensitivity using highly specific signal amplification systems as well as improved microscopy and optic devices as described recently (9,10). In addition to the HIV products, we are able to detect several cellular/molecular markers to analyze further viral trafficking, cellular activation, compartmentalization, and HIV interacting proteins including histone acetylates, apolipoproteins, and others (up to 5C6 colors). Our approach enables improved techniques of antigen recovery, staining, and confocal analysis resulting in outstanding identification and quantification of viral reservoirs. By using these Rabbit Polyclonal to C1QB methods, we are able to B-Raf-inhibitor 1 analyze millions of cells and focus only on the cells positive for viral HIV DNA/mRNA/protein using confocal microscopy, improved equipment, and imaging software. In this chapter, we will focus on 2 methods of detection of low levels of HIV proteins in cells. These methods can then be combined with assays for detection of HIV DNA and/or mRNA in the same samples, to obtain the most sensitive and reliable detection of viral reservoirs. Some of the technical improvements described here include: 1) to conserve antigens and nuclei acids during the processing of the sample even in archival materials; 2) using big pinholes to generate large optical sections to detect any positive signal; 3) for antibodies. 4) allows us to perform fast scanning of large areas in 3 dimensions to identify the few HIV-infected cells by 3D reconstructions and deconvolution; 5) to detect extremely narrow wavelengths and eliminates auto-fluorescence; 6) include cameras with recovery of 90% of photons per frame instead of the high resolution cameras for microscopy that only recover approximately 50% of photons and 7) Lastly, improved software and algorithms detect and quantify the signals generated by the different viral components (see details using other latent pathogens in (11,12)). The combination of all these factors enables us to detect, quantify, and localize specific signals from HIV reservoirs. 2. Materials 2.1 Tissue sections Any tissue section can be analyzed for viral reservoirs. The important point is preservation and size of the section (10C300 m) to allow analysis of millions of cells. Alcohol/Xylenes Phosphate buffered saline (PBS) and Tris buffered saline (TBS) Citrate Fish Gelatin Horse serum Sudan Black Sodium borohydrate Pontamine sky blue and 6.6-[(3, 3-dimethoxy[1,1-biphenyl]-4,4-diyl)bis(azo)]bis[4-aminuteso-5-hydroxy-1,3-naphthalenedisulfonic acid], tretrasodium Toluidine blue Triton-X Biotin blocking reagents Strepavidin conjugated to different fluorochromes or beads Alexa conjugated secondary antibody- Goat Anti-Rabbit IgG Prolong Gold anti-fade agent with DAPI 2.2. Leukocytes Whole blood or leukopacks from HIV infected or uninfected individuals. HIV-p24 ELISA (Perkin Elmer, Boston, MA; sensitivity: 12.5 pg/ml) or by COBAS Roche Amplicor v 1.5 (Roche, Germany; sensitivity 20 RNA copies/ml) to detect HIV infection. Lysis buffer Ficoll Paque plus Poly-lysine glass slides Phorbol myristate acetate (PMA) ACH-2 and OM-10 cell lines (13C16) Hela cells Paraformaldehyde (PFA) 3. Methods 3.1 Equipment Several types of confocal microscopes can be used depending on the brand. In our case we used an A1 Nikon confocal microscope with spectrum detection and unmixing separation systems. The configuration of the system is described in Figure 1. Using these configurations in addition to better protocols for staining and.