HAV 3A TM domain targets 3ABC to mitochondria and is essential for 3ABC cleavage of MAVS. Laser-scanning confocal microscopy images of transfected Huh7 cells showing cellular localization of (top frames) C terminally Flag-tagged HAV P3 expression products, (middle frames) mitochondria (MitoTracker), and (bottom frames) merged images with DAPI localization of nuclei. To compensate for quantitative differences in HAV protein expression, the green channel gain was increased for 3ABC-™ and decreased for 3ABC-C172A. Ectopically expressed HA-tagged 3ABC, 3ABC-C172A, and 3A, but not 3ABC- ™, associate with mitochondria. Cell fractions were prepared according to the scheme shown to the left, as described previously (5). CI-39, complex I 39-kDa subunit (mitochondria); PDI, protein disulfide isomerase (endoplasmic reticulum).
The colocalization of nucleolin, B23, and wt hepatitis B core antigen (HBcAg). Huh 7 cells were transiently transfected with pSVC plasmids. Upper left, DAPI (blue). Upper right, rabbit anti-HBcAg and FITC-labled goat anti-rabbit, green. Lower left, mouse anti-nucleolin and TRITC-labled goat anti-mouse. Lower right, the blue, green and red images were merged.
Monitoring the subcellular distribution of exogenously expressed viral proteins (mouse hepatitis virus (MHV)-A59 p28 protein). 17Cl-1 cells, cultured on chambered coverglasses, were transfected with pEGFP-pA59-p28 (Fig. 3a) and pEGFP-N1 (Fig. 3b). 24h after transfection, Hoechst 33342 was added to the culture medium, and live cells were observed with the LSM 510 UV META confocal microscope with appropriate filters for Hoechst 33342 (blue) and EGFP (green). Confocal images in the blue and green channels were merged to show the predominantly cytoplasmic distribution of p28-EGFP (Fig. 3a) and cytoplasmic/nuclear distribution of N1-EGFP. The graph below the images is illustrates the fluorescence intensity in the blue and green channels along the plain of the line (red) shown on each of the merged images.
Confluent human bronchial epithelial cells were infected with SARS-CoV at an MOI of 1.0 for 48-hrs before assessing the extent of viral infection using confocal microscopy. Briefly, paraformaldehyde-fixed and permeabilized infected Calu-3 cells were incubated with FITC-conjugated anti-SARS-CoV nucleocapsid protein (NP) and Texas red-conjuagted anti-F actin antibodies, according to the standard protocol. Cells were counterstained with antifade reagent containing DAPI. Fluorescent images were captured with a laser scanning confocal microcope (Zeiss LSM 510, Infectious Disease and Toxicology Optical Imaging Core facility at UTMB).
Three-color fluorescence analysis of the interaction of salmonella with intestinal epithelial cells. Cultured human intestinal epithelial cells, Caco-2BBe, were infected with Salmonella dublin expressing GFP-labeled flagellin (green) for 90 minutes. Cells were stained with DAPI (blue--Nucleus) and with Alexa Fluor 568 Phalloidin (red--Actin).
Two-color fluorescence analysis of the interaction of salmonella with epithelial cells. Forty sequential optical sections (z-sections) obtained imaging through the 100x/1.3 oil objective were compressed to one image (Fig. 2a). These stacked images were viewed from 32 angles of rotation around the z axis to provide a view from the x-axis (Fig. 2b). Cultured human intestinal cells, Caco-2BBe, were infected with Salmonella dublin expressing GFP-labeled flagellin (green) for 1.5 hours. Actin (red) was stained with Alexa Fluor 568 Phalloidin.
Wild type T. cruzi stained with syto-11, which stains DNA. Fluorescence image overlayed on the differential interference contrast image. Note fluorescence staining of the nucleus and kinetoplast. [Original image obtained with a C-apochromat 63x/1.2 W Cor]
Plasmodium gallinaceum ookinetes. Three-color fluorescence with antibodies specific for proteins of interest directly conjugated with Alexa-488 (green) and Alexa-594 (red). DNA was labeled with DAPI (Blue). The fluorescence images are shown with reference to the differential interference contrast image. [Original image obtained with a C-apochromat 63x/1.2 W Cor ]
Plasmodium gallinaceum ookinetes labeled with an antibody preparation directly conjugated with Alexa-594 (red). DNA was labeled with DAPI (blue). Fluorescence images merged with differential interferense contrast image. [Original image obtained with a C-apochromat 63x/1.2 W Cor]
Plasmodium gallinaceum ookinetes labeled with antibodies specific for proteins of interest directly conjugated with Alexa-488 (green) and Alexa-594 (red). DNA was labeled with DAPI (Blue). The differential intererense contrast image is shown in the lower left panel and the fluorescence and DIC images are merged in the lower right panel. Note membrane staining (red). [Original image obtained with a C-apochromat 63x/1.2 W Cor.]
Three-color confocal microscope image of a cytospin preparation of a human stem cell culture after 2 weeks of incubation in a Synthecon bioreactor using different concentrations of growth factors. CD34 phycoerytrin, blue; CD117 FITC-Ckit, green; and CD38 PerCP-red, red. Note that CD34 was detected at the cell membrane, where it had contact with other cells.
Imaging of telomeres on metaphase chromosome using a probe labeled with cy3 (excitation, 543nm; emission, 560-615 nm). The chromosomes were stained with DAPI (excitation, 351nm; emission 385-490 nm). Quantitative FISH, using the same labeling technique and interphase cells can be used to estimate telomere length.
Two-color fluorescence imaging was used to monitor the expression and cellular distribution of EGFP in stably transfected CHO cells. CHO cells (UV135) were transiently transfected with plasmid pEGFP-N3 vector (green--ClonTech) using LipofectAMINE Plus reagent (Gibco). The nucleus was stained with DAPI (blue). At 12 hr post-transfection, cells were fixed for 1 hr in reshly prepared 1% fparaformaldehyde (diluted in PBS at 4oC). Cells were air dried prior to mounting with Antifade solution (Molecular Probes).
Colocalization of an unspecified nonstructural viral protein (red) with a structural viral protein (green) in limited areas of the cytoplasm of Huh 7 cells. To better demonstrate the phenomena the image was cropped and enlarged successively (Fig. 1b, 2X; Fig. 1c, 4x). Note limited areas of yellow at the interface of the nonstructural and structural proteins.