Fluorescently labeled phalloidins have been extensively used to visualize the actin cytoskeleton; the dynamics of the actin cytoskeleton are important for a variety of cellular processes from cell motility to cell shape, from muscular contraction to cytokinesis, and more.
Molecular Probes (Invitrogen) is the leader in the field of fluorescently labeled compounds; among the compounds they offer are many fluorescent derivatives of phalloidin and phallacidin. They developed an entire range of labeled phalloidins with excitation wavelengths from 346 to 679 nm and emission maxima from 442 to 702 nm, respectively. The green-fluorescent dye – Alexa Fluor 488, the red-orange – Alexa Fluor 568, the red – Alexa Fluor 594, and the far-red – Alexa Fluor 647 conjugates are more fluorescent, photostable and resistant to photobleaching than the spectrally similar fluorescein, Lissamine, rhodamine B, Texas Red and Cy5 conjugates, respectively. Alexa Fluors 546, 568, 594 successfully replaced rhodamine phalloidin in many multicolor applications because their emission spectra are better separated from those of the green-fluorescent Alexa Fluor 488, Oregon Green and fluorescein dyes. The fluorescently labeled phalloidins are of the best quality and can be used to detect filamentous actin (F-actin), the polymerized form of actin. Labeled phallotoxins have a similar affinity for large and small actin filaments and bind in a stoichiometric ratio of one phallotoxin per actin subunit. They stain F-actin selectively at nanomolar concentrations and are used to identify and quantify actin in tissue sections, cell cultures or cell-free preparations. Phallotoxins have advantages over antibodies for actin labeling because their binding properties do not change appreciably with actin from different species and their nonspecific staining is negligible.
Molecular Probes has also reagents to detect monomeric, unpolimerized G-actin, and thus to investigate actin cytoskeleton dynamics in vivo. Using different fluorochrome probes for both monomeric and polymeric actin, one can simultaneously visualize these 2 actin pools and follow the kinetics of actin cytoskeleton.
Molecular Probes phallotoxins are bicyclic peptides isolated from the deadly mushroom Amanita phalloides. They are not cell permeant, therefore for living cells studies, they must be microinjected. As fluorescent phalloidin conjugates are not permeant to live cells, they can be used also to detect cells that have compromised membranes. In addition, Molecular Probes offers unlabeled phalloidin for blocking F-actin staining by labeled phallotoxins and for use as control.
We extensively use Rhodamine phalloidin and Alexa Fluor 488 phalloidin and the results are constantly spectacular. The stocks are reconstituted in methanol to a final concentration of 200 units/ml (6.6 µM) and are stable for at least 1 year at -20ºC. They are rather toxic and should be handled with care and protected from light in storage and during staining. We also keep aliquots at -100ºC. We usually use formaldehyde fixation with 16% formaldehyde in 1:1 PBS:heptane buffer as it best preserves actin in Drosophila embryos, the biological material we use. We dilute 5 µl of methanol stock solution in 200 µl PBS for each coverslip and use 2% BSA in PBS to reduce the background fluorescence. We then incubate for 1 h at room temperature or overnight at 4ºC. We perform the incubation in a moist chamber made from a Petri dish, wet Whatmann paper and Parafilm to avoid evaporation. We usually do triple labelings with Alexa 488, Alexa 568 and TOTO–3 iodide, also from Molecular Probes. We do not use any signal enhancer as the fluorochrome is extremely bright.
We capture images with a Nikon Eclipse TE2000-E inverted microscope (Nikon Mississauga, ON, Canada) equipped with a Perkin Elmer Life Sciences UltraVIEW confocal spinning disk (Perkin Elmer Life Sciences, Missisauga, ON, Canada) and a Hamamatsu Orca-ER camera (Hamamatsu Photonics, Hamamatsu, Japan).
To reduce photobleaching, we minimize the exposure of fluorescently labeled specimens to light with neutral density filters, expose to light only during image acquisition, use high numerical aperture objectives and low magnification. The photostability of fluorescent phalloidins can be considerably improved by mounting the stained samples with ProLong Antifade Kit or ProLong Gold Antifade Reagent from Molecular Probes. However, we use paraphenylendiamine in our mounting media, because it is cheaper to make it in Tris–glycerol than to buy the ready-made one.
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Dr. Rosalind Silverman
Postdoc
Department of Medical Genetics and Microbiology
University of Toronto
Canada
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