Rhodamine-phalloidin

Cleary AL. F-actin redistributions at the division site in living Tradescantia sto-matal complexes as revealed by microinjection of rhodamine-phalloidin. Protoplasma 1995 185 152-165. 

Fig. 3. Comparisons of wide-field (A) and confocal fluorescence images (B, mesoglea level C, apical) of rhodamine phalloidin-stained F-actin in a whole-mount hydra tentacle. The hydra was fixed and stained as described in Chapter 18. The bar represents 25 pm. All images were collected with a Nikon (New York) Microphot FX microscope (x40 objective lens). Confocal images were collected with the microscope connected to a Bio-Rad (Hercules, CA) MRC600 laser-scanning confocal system.

Fig. 5. Optical sectioning of rhodamine phalloidin-stained F-actin in a neutrophil migrating through a 5-pm pore of a polycarbonate membrane. The neutrophil migration is stimulated in response to 10 M Af-formytmethionyl-leucy 1-phenylalanine. (A), (B), and (C) correspond to O.S-pm optical sections indicated as sections A, B, and C, respectively, in Fig. 4. The bar represents 10 pm. The images were collected with a Nikon Microphot FX microscope (x60 Plan-apochromat lens, numerical aperture, 1.6) connected to a Bio-Rad MRC600 laser-scanning confocal system.

Nitrobenzoxydiazole (NBD) phallacidin or rhodamine phalloidin unconjugated phallacidin or phalloidin (Molecular Probes, Eugene, OR). Dissolve the phallotoxins in methanol to a final stock concentration of =3.3 pA/, and store at -20°C in the dark (see Note 1). 

The phallotoxins, phalloidin and phallacidin, are bicyclic peptides with mol wts of 789 and 847 Dalton, respectively. The NBD phallacidin and rhodamine phalloidin conjugates have been most frequently utilized in flow cytometric assays. The fluorescent phallotoxin conjugates have mol wts of 1000-1200 Dalton, are water-soluble, and stain actin at nanomolar concentrations (reviewed in ref. 19). Unconjugated phallotoxins should be obtained to verify the specificity of fluorescent phallotoxin staining to F-actin (see Note 4). 

Figure 9.4 Resveratrol induces morphological changes in cultured BPAEC. Panel A. Microscopic illustration of the stellar, cobblestone-like morphology characteristic of cultured normal BPAECs. Panel B. Illustration of elongated, spindle-shaped morphology characteristic of 100 pM resveratrol-treated cells. Cells are stained with rhodamine-phalloidin and viewed with a 20X objective. Note the long, tortuous projections.

Add to each reaction 50 pL of either rhodamine phalloidin or NBD-phal-lacidin stock solution (3.3 pM), mix the samples and incubate for 10 min at 37°C (see Note 4). 

C2 toxin-induced depolymerization of actin filaments is visualized by fluorescence labeling of actin with FITC- or Rhodamine-phalloidin. 

Deoxyribonucleic acid from calf thymus DNAse I solution FITC-phalloidin p-phenylendiamine rhodamine-phalloidin... 

A. Preparation of Rhodamine Phalloidin-Labeled Actin Filaments... 

FIGURE 2 Collage demonstrating the movement of rhodamine phalloidin-labeled actin filaments over a myosin-coated surface. Panels a-e are direct photographs of the video monitor at 30-sec intervals. Panel f shows a plot of the centroid position of selected actin filaments in the field, with arrows showing the direction of movement. The figure is taken from Collins et al. (1990). 

Two vol of 20 nM rhodamine phalloidin labeled actin in buffer B is used to wash the flow cell. 

Several tricks can be used to improve the quality of movement in the assay. One is to apply 2-3 vol of buffer A containing 5 pM of unlabeled actin and 1 mM ATP prior to step 4. After 1-2 min of incubation, the flow cell is washed with buffer A and then rhodamine phalloidin actin is applied as in step 4. This treatment appears to tie up the rigor heads with unlabeled (and therefore invisible) actin. The other treatment is to mix the myosin in buffer B prior to the start of an assay with a stoichiometric amount of actin in the presence of 5 mM MgCl2 and 5 mM ATP, followed by centrifugation in a TLIOO ultracentrigue for 10 min at 100,000g, which will pellet the actin and attached rigorlike heads, but leave active myosin in the supernatant. Finally, the inclusion of tropomyosin (40-100 nM) in the motility buffer increases not only the rate of movement but also the quality of movement (Umemoto and Sellers, 1990). 

FIGURE 3 Construction of the flow cell for the sliding actin in vitro motility assay. The paired irregular yellow strips represent the grease that adheres the coverslip (on top) to the slide. The coverslip slivers that are used as spacers are narrower than the top coverslip. The pink liquid represents a solution of rhodamine phalloidin-labeled actin that is being applied to the flow cell. 

Fig. 13 Fluorescent Images and the corresponding line profiles of the F-actin fibers red) of ADSCs seeded on (a) glass surface and (b) silk fibroin/chitosan (SFCS) scaffold. F-actin fiber density of ADSCs was quantified and confirmed by line-profile analysis of the fibers using Image software. The x-axis is the distance in microns, and the peaks correspond to the intensity of the rhodamine-phalloidin stain (red), whose peak maximum occurs at the location of the fibers along the line. Nuclei were stained with DAPI (blue) [162]...

Wash tJae cells twice with PBS, then incubate with secondary antibody and rhodamine-phalloidin for 30 min. For negative control staining, use IgG or the secondary antibodies only. 

Staining the Actin Cytoskeleton 1. Intracellular buffer (2x). 280 mM KCl, 2 mM MgCl, 4 mM EGTA, 40 mM Hepes of pH 7.5, 0.4% low endotoxin albumin from human serum (Sigma) see Note 5). 2. Fixation buffer (2x). 640 mM sucrose, 7.4% formaldehyde (Sigma) in 2x intracellular buffer store at 4°C (tee Note 6). 3. Stain buffer. 0.2% triton, 2 pl/ml rhodamine phalloidin (Invitrogen) in lx intracellular buffer (rccNote 7). 

Unlike Alexa Fluor 594, rhodamine phalloidin increases its fluorescence upon binding to actin filaments. This makes it preferable to Alexa Fluor 594. However, for imaging in the green channel, we would use Alexa Fluor 488 or similar fluor-ophore over the rapidly bleaching FITC. 

Fig. 3 Actin cytoskeleton and focal adhesion. F-actins were visualized using rhodamine phalloidin (red), and vinculins found in focal adhesions were stained using fluorescently conjugated antibodies. (A) Immunofluorescent images of a thin filamentous actin meshwork and vinculins showed that osteoblasts contain fewer and smaller focal adhesions. (B) In contrast, hMSCs showed thick actin stress fibers, and multiple and large adhesion contacts.

Rhodamine-phalloidin-labeled cytoskeletal actin cell, dried on silica 34... 

Figure 13.4 Details of fluorescence micrographs of the textile samples with endotheUal cells dyed with Rhodamine Phalloidin and DAPl, after 5 days of proliferation on the hiomaterial woven/velour sample. Slide (a) shows a representative plain woven section of the prosthesis, where few cells have proliferated. Slide (b) shows a representative velour section where endothelial cells have proliferated only along the fibers, with very few coimections between fibers.

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