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Fluorescence Digital Image Gallery

African Water Mongoose Skin Fibroblast Cells (APM Line)

The APM cell line was established at The Naval Biosciences Laboratory (NBL) in Oakland, California from the skin of an African water mongoose (Atilax paludinosus). An elusive animal, the African water mongoose exhibits solitary, nocturnal habits and is a good swimmer, although it frequently lingers in shallow waters where it catches shellfish, crabs, frogs, and similar aquatic and semi-aquatic animals for food.

APM mongoose cells exhibit fibroblast morphology and, similar to other fibroblast lines, are among the easiest cells to grow in culture. Cell biologists hypothesize that the ability of fibroblasts to grow so readily outside of the body is associated with their central role in the healing of wounds, which necessitates their proliferation when confronted with injury or other less than optimal conditions. Fibroblasts are also generally considered to exhibit relatively solitary lifestyles, which some have suggested may also be a factor in their favorable growth in culture.

Fibroblasts are the least specialized cells of the connective tissue and are capable of differentiating into an array of different mature cell types, including bone cells, fat cells (adipocytes), smooth muscle cells, and cartilage cells (chondrocytes). Several different dynamic factors are involved in the regulation of fibroblast transformations, such as hormones, growth factors, and the makeup of the extracellular matrix, to which the cells contribute through secretions. Also, some studies indicate that fibroblasts present in various locations throughout the body are inherently different and that even fibroblasts inhabiting a single area may possess some important characteristics that affect their differentiation capabilities. Indeed, fibroblasts derived from different tissues also often exhibit varying characteristics in culture. For instance, fibroblasts from the skin, similar to those of the APM line, do not tend to exhibit as much flexibility in differentiation as do fibroblasts derived from bone marrow, though they can be provoked to change their character under certain conditions.

In a double immunofluorescence experiment, the adherent monolayer culture of African water mongoose cells illustrated above was fixed, permeabilized, blocked with 10-percent normal goat serum, and then treated with a cocktail of mouse anti-NPCP (nuclear pore complex protein) and rabbit anti-giantin (Golgi complex) primary antibodies followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Alexa Fluor 488 and Alexa Fluor 568, respectively. The filamentous actin network was counterstained with Alexa Fluor 350 conjugated to phalloidin. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.

Additional Fluorescence Images of African Water Mongoose (APM) Cells

Visualizing the Microtubule and Filamentous Actin Networks in APM Cells - The microtubule network from the adherent culture of APM fibroblast cells presented in this section was immunofluorescently labeled with anti-tubulin mouse monoclonal primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 568 (yielding red emission). In addition, the cells were labeled for cytoskeletal F-actin with Alexa Fluor 350 (blue emission) conjugated to phalloidin, and for cell nuclei with SYTOX Green, a classic nucleic acid stain.

Focal Adhesions in African Water Mongoose Skin Fibroblast Cell Cultures - The adherent culture of APM cells featured in this section was immunofluorescently labeled with anti-vinculin mouse monoclonal primary antibodies followed by goat anti-mouse IgG secondary antibodies conjugated to Texas Red. In addition, the specimen was simultaneously stained for DNA with the ultraviolet-absorbing probe Hoechst 33258, and for the cytoskeletal filamentous actin network with Alexa Fluor 488 conjugated to phalloidin.

Proximity of the Golgi Complex and Nucleus in APM Monolayer Cell Cultures - The close proximity between the Golgi complex and nuclei in African water mongoose cells was probed in a double immunofluorescence experiment with mouse anti-NPCP (nuclear pore complex protein) and rabbit anti-giantin primary antibodies. The antibody targets were visualized with goat secondary antibodies conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively, while the actin cytoskeletal framework was labeled with Alexa Fluor 350 conjugated to phalloidin.

African Water Mongoose Fibroblasts with MitoTracker Red CMXRos, Alexa Fluor 488, and Hoechst 33258 - Using a popular triple-fluorophore staining technique for mitochondria, filamentous actin, and the nucleus, a log phase monolayer culture of APM cells was first treated with MitoTracker Red CMXRos for one hour, and then fixed with medium containing 3.7-percent paraformaldehyde. After permeabilization and blocking with bovine serum albumen, the cells were labeled with Alexa Fluor 488 conjugated to phalloidin and counterstained with Hoechst 33258.

Histone and Peroxisome Distribution in APM Cell Cultures - In a double immunofluorescence experiment, an adherent monolayer culture of African water mongoose fibroblast cells was fixed, permeabilized, blocked with 10 percent normal goat serum, and treated with a cocktail of mouse anti-histones (pan) and rabbit anti-PMP 70 (peroxisomal membrane protein) primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies (IgG) conjugated to Texas Red and Alexa Fluor 488, respectively. The filamentous actin network was counterstained with Alexa Fluor 350 conjugated to phalloidin.

Localizing the Tubulin and Filamentous Actin Networks in African Water Mongoose Cells - Immunofluorescence with mouse anti-alpha-tubulin was employed to visualize distribution of the microtubule network in a log phase monolayer culture of African water mongoose skin cells. The secondary antibody (goat anti-mouse IgG) was conjugated to Alexa Fluor 568 and mixed with Alexa Fluor 488 conjugated to phalloidin to simultaneously image tubulin and the actin cytoskeleton. Nuclei were counterstained with Hoechst 33258.

APM Monolayer Cultures with Alexa Fluor 594, Oregon Green 488, and Hoechst 33258 - In a triple-fluorophore experiment similar to the one described in the previous section, a culture of African water mongoose cells was fixed, permeabilized, blocked with 10-percent normal goat serum in PBS, and then treated with mouse anti-alpha-tubulin primary antibodies. The secondary antibody (goat anti-mouse IgG) was conjugated to Oregon Green 488 and mixed with Alexa Fluor 594 conjugated to phalloidin. Nuclei were again counterstained with Hoechst 33258.

Localization of the Golgi Complex and Nuclear Pore Complex Proteins in APM Cell Cultures - Using double immunofluorescence, an adherent culture of APM cells was washed, fixed, permeabilized, blocked with 10-percent normal goat serum, and treated with a cocktail of rabbit anti-giantin (Golgi complex) and mouse anti-NPCP (nuclear pore complex proteins) primary antibodies. The target antigens were visualized with goat anti-rabbit and anti-mouse secondary antibodies (IgG) conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively. The filamentous actin cytoskeletal architecture was labeled with Alexa Fluor 350 conjugated to phalloidin.

African Water Mongoose Fibroblast Cells with Texas Red-X, Oregon Green 488, and Hoechst 33258 - In order to label the intermediate filaments in a log phase adherent APM culture, the fixed and permeabilized cells were blocked and treated with mouse anti-vimentin (porcine eye lens) primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Texas Red-X. Filamentous actin was visualized with phalloidin conjugated to Oregon Green 488, while the nuclei were stained with Hoechst 33258.

Triple Fluorophore Labeling of APM Cells with MitoTracker Red CMXRos, BODIPY FL, and Hoechst 33258 - A log phase monolayer culture of African water mongoose cells was treated with MitoTracker Red CMXRos in growth medium for one hour, washed, and fixed with 3.7-percent paraformaldehyde in medium containing serum. After washing and permeabilization, the cells were blocked with bovine serum albumen in PBS and labeled with BODIPY FL conjugated to phallacidin. The nuclei were subsequently counterstained with Hoechst 33258.

Targeting Peroxisomes and Clathrin Proteins in African Water Mongoose Cells with Immunofluorescence - In this section, the featured culture of APM cells was immunofluorescently labeled with primary anti-clathrin (heavy chain) mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Texas Red in order to target the cytoskeletal network. In addition, peroxisomes present in the culture were simultaneously labeled with Oregon Green 488 conjugated to goat secondary antibodies directed against rabbit anti-PMP 70 (peroxisomal membrane protein 70) primary antibodies. Nuclei were counterstained with Hoechst 33258.

The Distribution of Nonmuscle Myosin II and Filamentous Actin in APM Cell Cultures - A fixed and permeabilized monolayer culture of APM cells was treated with mouse anti-nonmuscle myosin II (heavy chain) monoclonal primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Texas Red. Oregon Green 488 conjugated to phalloidin was included in the secondary cocktail to target the filamentous actin network. After the immunofluorescence and phallotoxin labeling, the cells were washed and counterstained with Hoechst 33258.

The Microtubule and Actin Cytoskeletal Networks in African Water Mongoose Fibroblast Cells - After fixation in 0.3-percent glutaraldehyde and permeabilization with Triton X-100, an adherent culture of APM cells was treated with mouse anti-alpha-tubulin monoclonal primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Oregon Green 488. Mixed together with the secondary antibody was a phalloidin conjugate of Alexa Fluor 594, targeting the filamentous actin network. Nuclei were visualized by staining with Hoechst 33258 in Hanks' buffer for 30 minutes.

Localizing the Mitochondria, Filamentous Actin, and Nuclei in APM Fibroblast Cell Cultures - The popular multiple fluorophore combination of MitoTracker Red CMXRos, Alexa Fluor 488 conjugated to phalloidin, and Hoechst 33258 was employed to perform a triple label experiment on a monolayer of African water mongoose cells. The trio of fluorophores stains the cells with red (mitochondria), green (filamentous actin), and blue (DNA in the nucleus) fluorescence emission.

Examining the Proximity Between the Golgi Complex and Nucleus in African Water Mongoose Cells - In a manner similar to that described above, the close proximity between the Golgi complex and nuclei in African water mongoose cells was probed in a double immunofluorescence experiment with mouse anti-NPCP (nuclear pore complex protein) and rabbit anti-giantin primary antibodies. The antibody targets were visualized with goat secondary antibodies conjugated to Alexa Fluor 568 and Alexa Fluor 488, respectively, while the actin cytoskeletal framework was labeled with Alexa Fluor 350 conjugated to phalloidin.

APM Monolayer Cell Cultures with Texas Red-X, Alexa Fluor 488, and Hoechst 33258 - A goat anti-mouse secondary antibody conjugate of Texas Red-X was employed to visualize mouse anti-alpha-tubulin (IgG) primary antibodies in an adherent culture of African water mongoose cells. In addition, the cytoskeletal architecture was imaged with Alexa Fluor 488 conjugated to phalloidin, and DNA in the nucleus was targeted with the bis-benzimide fluorophore, Hoechst 33258.

SYTOX Green, Alexa Fluor 350, and Alexa Fluor 568 Fluorescence in African Water Mongoose Cells - Analogous to the digital image presented in the link above, a goat anti-mouse secondary antibody conjugate of Alexa Fluor 568 was employed to visualize mouse anti-alpha-tubulin (IgG) primary antibodies in an adherent culture of African water mongoose cells. In addition, the cytoskeletal architecture was imaged with Alexa Fluor 350 conjugated to phalloidin, and DNA in the nucleus was targeted with SYTOX Green.

Visualizing the Vimentin Intermediate Filament Network in APM Cell Cultures - A fixed and permeabilized culture of adherent APM cells grown on cover glasses was treated with mouse anti-vimentin primary antibodies followed by goat anti-mouse secondary antibodies (IgG) conjugated to Texas Red-X. The filamentous actin network and DNA in the nuclei were simultaneously imaged by labeling with Oregon Green 488 conjugated to phalloidin and Hoechst 33258, respectively.

Triple Fluorophore Staining Patterns in African Water Mongoose Cell Cultures - Applying a collection of popular mitochondrial, actin, and DNA probes, a culture of APM cells was grown to log phase, treated with MitoTracker Red CMXRos before fixing, and then labeled with phalloidin and Hoechst 33258 after permeabilization. The red fluorescence arises from the mitochondrial dye (MitoTracker), while the phalloidin was conjugated to Alexa Fluor 488 to generate green fluorescence. Nuclei are rendered in light blue.

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