CST, 8953S, Alexa Fluor® 555 Phalloidin

Cellular Dyes for studying in the research area. Product Usage Information To make a 100X stock, reconstitute the lyophilized material in 100 µl anhydrous DMSO. Fix cells for 15 minutes using fresh, methanol-free 4% formaldehyde, then rinse three times in PBS for 5 minutes each. Once fixed, please follow CST protocol for immunostaining. Following incubation of the primary and secondary antibodies, Alexa Fluor ® 555 Phalloidin can be diluted 1:100 (1 μl per 100 μl assay volume) in PBS and added to the cells. Allow to incubate for 15 minutes at room temperature, then rinse once with PBS. Coverslip slides with ProLong Gold Antifade Reagent and examine specimen using appropriate excitation wavelength. Note: Product usage information for this product has changed effective September 01 2024. Previous recommendations were to use either methanol or DMSO as a solvent for lyophilization of the material; It is now recommended to ONLY use DMSO. Storage This material is provided as lyophilized solid that is stable for 1 year at -20°C, desiccated and protected from light. Phalloidin conjugates should be reconstituted in anhydrous DMSO to make stock solutions, please refer to the directions for use for details. Once reconstituted in DMSO, stock solutions are stable for 1 year at -20°C. Stability in aqueous solutions is low and the conjugate should only be in the presence of an aqueous solution during incubation with cells. Background Actin, a ubiquitous eukaryotic protein, is the major component of the cytoskeleton. At least six isoforms are known in mammals. Nonmuscle β- and γ-actin, also known as cytoplasmic actin, are predominantly expressed in nonmuscle cells, controlling cell structure and motility (1). Actin exists mainly as a fibrous polymer, F-actin. In response to cytoskeletal reorganizing signals during processes such as cytokinesis, endocytosis, or stress, cofilin promotes fragmentation and depolymerization of F-actin, resulting in an increase in the monomeric globular form, G-actin (2). Phalloidin is a natually occuring toxic bicyclic peptide found in the deathcap toadstool, Amanita phalloides, that rapidly binds to F-actin with strong affinity (3). Alternate Names Alexa Fluor Phalloidin; lexa Fluor 555 Phalloidin; Phalloidin