1. Volume 137, Issue 4, Pages 1448-1458 (October 2009)
Visualizing Hepatitis C Virus Infections in Human Liver by Two-Photon Microscopy 
Yuqiong Liang, Tuya Shilagard, Shu–Yuan Xiao, Ned Snyder, Daryl Lau, Luca Cicalese, Heidi Weiss, Gracie Vargas, Stanley M. Lemon 
Gastroenterology 
Volume 137, Issue 4, Pages (October 2009)
DOI: /j.gastro
Copyright © 2009 AGA Institute Terms and Conditions

2. Figure 1
2PE detection of NS3 antigen in HCV-infected cells by indirect immunofluorescence using a Qdot anti-Ig conjugate. (A and B) Conventional laser scanning confocal microscopic images of sections of paraffin-embedded, formalin-fixed liver tissue obtained by percutaneous biopsy from (A) an HCV-negative patient with nonalcoholic steatohepatitis or (B) a treatment-naive, chronic hepatitis C patient with stage 3 fibrosis and serum HCV-RNA level of 724,114 IU. After antigen retrieval (see Materials and Methods section), tissue sections were labeled with anti-NS3 mAb followed by a fluorescein isothiocyanate–anti-Ig conjugate. Fluorescence (green) was visualized by excitation at 488 nm with an emission bandpass filter of 505–530 nm. Bar, 50 μm (20× objective). (C) 2PE microscopic image of Huh7 cells 2 days after infection with JFH-1 strain HCV. After fixation, cells were labeled with anti-NS3 mAb, followed by goat anti-mouse Ig Qdot-565 conjugate. Qdot fluorescence (red) was visualized by excitation at 800 nm with an emission bandpass filter of 555–575 nm. Bar, 50 μm (40× objective, panels C–H). (D) 2PE image in panel C merged with bright field image. (E and G) 2PE images of HCV-infected human liver (section from the same tissue block as panel B). After antigen-retrieval, cells were labeled and Qdot fluorescence was visualized as in panel C. Clusters of HCV-infected cells with cytoplasmic staining for NS3 (red) are delineated against a background of negative hepatocytes. (F and H) Images from panels E and G merged with related bright field images.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Specificity of 2PE detection of HCV core antigen in frozen sections of human liver using a direct anti-core mAb (C7-50) Qdot-655 conjugate. (A) Top panel: 2PE image of cirrhotic liver from an HCV-infected patient (H5, see Table 1) undergoing resection of HCC. Serum HCV-RNA level (genotype 3) was 335,556 IU. Core antigen is present in the cytoplasm of numerous cells. Bottom panel: Merged 2PE images obtained with the anti-core Qdot conjugate (excitation, 800 nm; emission bandpass filter, 645–665 nm; green) and DAPI (excitation, 800 nm; emission bandpass filter, 435–485 nm; blue). Bar, 50 μm (40× objective). (B) Top panel: Twenty systematically collected 2PE microscopic fields (merged anti-core Qdot-655 conjugate and DAPI images) of a section of liver from patient H5 that was preblocked with a nonspecific (anti-HAV) mAb (K24F2) before labeling (see Materials and Methods section). Of 3457 cells present in all 20 fields, 277 (8.01%) were considered positive for HCV core antigen. Bottom panel: A similar set of 20 systematically collected 2PE images from the adjacent liver section, 12 μ distant from that shown in the top panel. This section was preblocked with anti-core mAb before labeling with the anti-core Qdot-655 conjugate. Of 3822 cells counted in all 20 fields, only 22 (0.58%) were considered positive for HCV core antigen, a 93% reduction from the images shown in the top panel, supporting the specificity of the labeling. Bar, 100 μm (40× objective). (C) Percentage of cells (in 20 systematically collected field images) that were positive for HCV core antigen after preblocking with nonspecific vs specific antibodies in sections of liver from patients H1, H5, and H9.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
Tissue colocalization of HCV core and NS3 antigens. (A) 2PE images of cultured cells conditionally expressing HCV core (top panels, Huh7-191/20 cells) and NS3-4A (bottom panels, UNS3-4A-24 cells) under control of the Tet-Off promoter (Clontech, Mountainview, CA). Cells were grown in the presence or absence of tetracycline (2 μg/mL), then fixed and dually labeled for NS3 antigen with anti-NS3 mAb (primary) and an anti-mouse Ig Qdot-605 conjugate (detected with excitation at 800 nm and an emission band pass filter of 595–615 nm) (red), followed by direct labeling with the anti-core Qdot-655 conjugate and 2PE detection as in Figure 2 (green). Nuclei are counterstained with DAPI (blue). Bar, 50 μm (40× objective). (B) Similar dual labeling of liver tissue from patient H1 with anti-NS3 mAb (primary) and anti-Ig Qdot-565 conjugate (middle panel, red), followed by direct labeling with anti-core Qdot-655 (left panel, green). Bar, 50 μm (40× objective).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
2PE detection of dsRNA in HCV-infected liver. (A) Laser-scanning confocal microscopy of HJ3-5 HCV-infected cells labeled with rabbit anti-NS5A (detection with anti-rabbit Ig Alexa-594, green) and mouse anti-dsRNA (anti-mouse Ig Alexa-488, red). Nuclei were counterstained with DAPI. Bar, 20 μm (63× objective). For details, see the Supplementary Materials and Methods section. (B) 2PE images of frozen liver sections from patients H5 (top) and H7 (bottom) that were dually labeled with anti-dsRNA and anti-Ig Qdot-565, followed by anti-core Qdot-655 conjugate. Bar, 50 μm (40× objective). In both panels, arrows indicate examples of cells with abundant core but little detectable dsRNA that are likely to have been infected recently.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
HCC core antigen expression in cirrhotic and HCC tissue. (A) Percentage of cells containing detectable HCV core antigen in frozen liver sections. A low percentage of cells (0.37% ± 0.18%) in 3 control tissues (C1–C3) represent background labeling. Core antigen was detected in all 9 HCV-infected tissues (H1–H9, 1.7%–21.6% of cells). Two tissue samples were examined from patient H9 who had a serum HCV-RNA level of less than 50 IU: one close to (H9p) and the other distant from (H9p) a resected HCC. An average of 4090 ± 669 cells (DAPI-stained nuclei) were screened for core antigen in each tissue section. (B) Cumulative HCV core antigen field labeling scores in 9 HCV-infected (gray lines) and 3 control (red lines) liver tissue samples. The percentage of cells labeled with anti-core antibody in each of 20 systematically sampled microscopic fields was added cumulatively to generate a linear plot reflecting the distribution of HCV infection. Horizontal segments indicate regions of the tissue section in which no positive cells were detected. Patient number and serum HCV-RNA results are shown on the right. (C) 2PE images of core protein expression in tissues from patient H9 (serum HCV RNA level, <50 IU) collected close to (H9p, frames i–ii) or distant from (H9d, frames iii–iv) HCC. Frames i and iii show cells labeled directly with the anti-core Qdot-655 conjugate; these images are merged with DAPI staining in frames ii and iv. Frames v and vi show HCV core and dsRNA labeling, respectively, in a section of tissue taken from a site close to the tumor. Bar, 50 μm (40× objective).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
Dual labeling of frozen tissues from patients with HCV infection with anti-elastin antibody and anti-core Qdot-655. (A) Clusters of HCV-infected cells adjacent to fibrous septae in 2 cirrhotic patients, H6 (frames i and ii) and H8 (frames iii and iv). Frames i and iii show elastin (red) under low magnification. Bar, 200 μm (10×/0.3 objective, 2PE not available). Frames ii and iv show DAPI-labeled nuclei (blue) and core detected by 2PE excitation (green) and elastin (red) detected by 488 nm excitation at higher magnification (field identical to the yellow box in low-power view). The arrow indicates a collection of inflammatory cells infiltrating the fibrous septae. Bar represents 50 μm (40×/0.8 W objective). (D) 2PE detection of core expression in a regenerating liver nodule (patient H2). Frames i and ii show images collected with a 10× and 40× objective (see legend to panel A). Frame iii shows H&E staining of the adjacent 12 μ section revealing extensive well-differentiated HCC. Bar, 400 μm (10× objective).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions