Phalloidin actin stain

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

Drosophila embryos are protected both by an outer layer called chorion and an impermeable and opaque vitelline membrane. Therefore preparation of whole mount Drosophila embryos for staining with antibodies and/or other fluorescent markers must go through the following steps chorion removal, fixation, vitelline membrane removal, and membrane permeabilization. The next subsection introduces the basic procedures for embryo collection and chorion removal that are common to all protocols described here, as well as the two most common fixation methods with or without methanol (the latter requiring hand devitellinization of embryos). The first one works well for immunostaining, while the second is ideal for F-actin staining with phalloidin. 

F-actin staining using FlTC-labeled phalloidin is carried out using 1-2 X 10 cells see Note 6). Stimulated cells are pelleted in a microcentrifiige at 300 x [ for 2 min. The supernatant is removed and cells are fixed and permeabilized with 400 pi of BD Cytoperm/Cytofix buffer for 20 min at room temperature see Note 7). Cells are washed with 2 ml cold BD Perm/Wash buffer twice, pelleted at 500 x for 5 min at4°C, followed by staining in the residual BD Perm/Wash buffer (approximately 100-150 pi) with 5 pi of 0.3 mM FITC-labeled phalloidin for 30 min on ice in the dark. 

Fig. 3. HIV-1 envelope signaling triggers actin rearrangement. Resting CD4T cells were treated with a laboratory-adapted viral strain, HIV-1 4.3 (a), or with a primary viral isolate, HIV-1 g3ug 4e (b), or with gpl 20IIIB (100 pM) (c) for various times, fixed and permeabilized, and then stained with FITC-phalloidin for F-actin and analyzed by flow cytometry. Shown are histograms of F-actin staining.

Fig. 3 Cell spreading of RAW 264.7 macrophages cultured on polyacrylamide gel surfaces with varying stiffness. Differential interference contrast microscopy images (A) and con-focal images for actin staining (B) from RAW 264.7 macrophages on a less rigid (1.2 kPa) versus rigid (150 kPa) polyacrylamide gel coated with poly-L-lysine. Green color represents actin staining (phalloidin) with blue stained nuclei (Hoechst). Adapted from [39] with permission.

Testis squashes can be stained with antibodies directed to specific proteins, with rho-damine-phalloidin to visualize F actin, and with DNA-binding dyes. Best results are obtained when these stainings are carried out in the following order immunostaining, F-actin staining, and DNA staining. This implies that multiply stained preparations are all mounted in the media described for DNA staining (see Protocol 5.8, step 2). 

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. 

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. 

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.

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)...

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... 

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.)... 

After reducing free aldehyde groups by treatment with sodium borohydride (1 mg/ml in PBS for 10 min), cells are stained for filamentous actin by incubating for 30 min with 0.1 (xg/ml tetrame-thylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma P1951) in PBS. 

The cytoskeleton is a cellular scaffold within the cytoplasm of the cell or within structures such as flagella, cilia, and lamellipodia. The cytoskeleton plays a crucial role in cellular integrity/structural support, cell division, cell motility, and intracellular transport. Staining of the cytoskeleton may be achieved by immunofluorescence in fixed cells however there are also dyes available, such as fluorescently labelled phalloidin, which directly labels actin filaments, as well as tubulin tracker which labels microtubules (Molecular Probes). Phalloidin is poorly permeable to living cells however, phalloidin derivatives with improved permeability properties are also available. Although both phalloidin derivatives and microtubule tracker can be used in live cells, it should be noted that both dyes are toxic as they inhibit cell division, and therefore limits their applications. 

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