Phalloidin staining

Doris FP, Steer MW. Effects of fixatives and permeabilisation buffers on pollen tubes implications for localisation of actin microfilaments using phalloidin staining, Protoplasma, 1996 195 25-36. 

Apply the phalloidin-staining solution for 20 min at room temperature (generally, any temperature between 4°C and 37°C is suitable). 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

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.

Other assays are being explored in HTS platforms to identify potential inhibitors of invasion, for example, compounds that affect the shape or morphology of cells or their ability to generate invadopodia. Quintavalle et al. recently described such a method initially using Src-transformed NIH 3T3 fibroblasts grown on 384-well optical plates. Imaging of cellular and nuclear morphology combined with phalloidin-stained F-actin was used to discriminate compounds which reduced (or enhanced) the number of... 

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]...

Figure 9.3 Distribution of focal adhesion proteins on RGD nanopattemed surfaces. Embryonic rat fibroblasts were seeded onto nanopattemed surfaces with either a 58- or 108-nm separation distance between RGD ligands. The localization of focal adhesion proteins vincuhn and paxillin were visualized using immunofluorescence at 3 and 24 h post-seeding. Cells seeded on the 58-nm nanopattemed surfaces showed significantly higher degrees of cell spreading compared with those on the 108-nm surfaces. Actin filaments in the fibroblasts were stained using an immunofluorescent phalloidin stain [64],...

Cytokinesis (cell division) in animal cells involves the progressive formation in telophase of a furrow between the two daughter cells in the equator of the mitotic spindle. Immunofluorescent staining of the cortical cytoplasm at the site of the contraction ring reveals an abundance of actin as well as myosin, a-actinin, and filamin (Fishkind and Wang, 1995). Cytokinesis is highly sensitive to actin-myosin inhibitors such as cytochalasin and phalloidin. 

Fig. 20.1. Confocal images of whole mounts of the ovijector region of A suum stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to show muscle and with an anti-RFamide antiserum coupled to fluorescein isothiocyanate (FITC) to show FaRPergic nerves. (A) Main ventral nerve cord encircles opening of ovijector where it meets the body wall and is immunopositive for FaRPs. (B) Flat-fixed preparation of the ovijector showing circular muscles and tracts of parallel FaRPergic nerves (arrows). (C) Detail of the circular muscle of ovijector and associated nerves (arrows). (D) A FaRPergic cell body is localized in the ventral nerve cord at junction with ovijector and provides innervation to ovijector muscle.

When staining with any of the fluorescent phalloidins, dilute 10 pL methanolic stock solution into 400 pi PBS. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution. It may also be useful to preincubate fixed cells with PBS containing 1 % BSA. 

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). 

Fig. 1 Lateral line neuromasts in zebrafish larvae, (a) Lateral view of live untreated 8 dpf zebrafish larvae (anterior to the left and dorsal up) stained with 5 pM 4-Di-2-Asp. (b) A neuromast from a fixed 5 dpf eleutheroembryo containing hair cells labeled for f-actin with phalloidin-Alexa 488. (c) Lateral view of a fixed 5 dpf larva containing lateral line neuromast stained with anti-acetylated alpha-tubulin, (d) Hair cells in a neuromast, with the stereocilia bundles in green (phalloidin-Alexa 488) and the kinocilia in red (alpha-tubulin, 1/1000)...

To study the implications of spectral phase correction for two-photon depth-resolved imaging, we imaged a thick section of monse kidney tissne, stained with DAPl (cell nnclei), Mitotracker-488 (mitochondria), and Phalloidin-568 (actin). The resnlts, snmmarized in Fignre 8.5, demonstrate increased penetration depth when higher-order dispersion is compensated via MllPS, compared to GDD-only compensation. The images show the collagen wall components of a blood vessel in the monse kidney at a depth of 40 am. The collection dnct region above it, at a depth of 50 jam, is seen only when MllPS is applied. 

Selective two-photon excitation can provide additional contrast for single-cell imaging. It might be particularly useful for co-localization studies. In Figure 8.8, it is illustrated on HeLa cells. The cells were co-stained with Phalloidin-350 (actin filaments) and with MitoTracker 488 (mitochondria). An amplitude shaping was used... 

FIGURE 8.8 A section of a single HeLa (human cervical cancer) cell stained with Phalloidin 350 (actin filaments) and MitoTracker488 (mitochondria) acquired using selective two-photon excitation, without fluorescence color filters. The image size is about 25 (xm. (From Dantus, M., Lozovoy, V. V., and Pastirk, I. Laser Focus World, 43(5) 101-104. 2007. Used with permission.)... 

Figurel9.1 Confocal imagesofhuman hepatocyteson PEEK-WC-PU membranes byactin staining with FITC-phalloidin (green) and by nucleic acid staining with DAPI (blue). Scale bar 10pm.

Alexa Fluor 543-conjugated Phalloidin actin stain, diluted in methanol to 200 U/mL according to the instructions given by the manufacturer (Invitrogen). 

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.

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