Phalloidin preparation

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.

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. 

Fig. 1. Examples of fluorescence preparations of Drosophila whole mounts using the protocols is described in this chapter. All confocal images were obtained with a LeicaTCS4D confocal microscope, (a) Confocal optical section of a D. melanogaster embryo whole mount at blastoderm stage double stained with phalloidin—rhodamine (red) and DAPI (blue) to allow simultaneous visualization of nuclei and cortical actin around cell membranes. Anterior is to the left.

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

Similarly a thioether was first prepared in the synthesis of the toxic mushroom peptide phalloidin and the homodetic ring was closed only subsequently... 

Cells were washed with prewarmed PBS (Fluka Chemicals, Buchs, Switzerland) and fixed in a 4% paraformaldehyde solution (freshly prepared from a 20% stock solution, paraformaldehyde powder from Fluka Chemicals, Buchs, Switzerland). After rinsing with PBS, the cells were penneabilized using 0.5% Triton X-100 (Fluka Chemicals, Buchs, Switzerland). The substrata were incubated with Alexa Fluor 488 phalloidin (1 100 dilution in PBS Invitrogen AG, Basel Switzerland) for 25 min at room temperature and rinsed with PBS. The nucleus was stained with DAPI (1 1000 dilution in PBS Invitrogen, Basel, Switzerland) for 15 min at room temperature. The substrata were rinsed again with PBS and kept in PBS in order not to dry off. 

Preparaiion. The following data were obtained from an actual preparation of phalloidin (396). Two hundred twenty kilograms of fresh fungi (Amanita phalloides)... 

The purity of phaUoidin may be checked as follows to 1 drop of a concentrated solution of phalloidin are added 20 drops of concentrated sulfuric acid containing Fe(III). After several minutes, a stable blue color appears. K the preparation contains a- or /3-amanitin, the color will be more or less green. 

It has a characteristic ultraviolet absorption spectrum in aqueous solution (535,628). In water, phalloidin gives a neutral solution. Various derivatives can be prepared from it, in particular an acetyl derivative crystallizing as prisms from aqueous acetone, and melting with decomposition at 203-205° C. 

Sorm and Keil (535) recently studied the structure of phalloidin in a preparation whose homogeneity had been checked by partition chromatography on starch and by ionophoresis. The ultraviolet spectrum was identical to that determined by Wieland (628). The total hydrolysis of this phalloidin yielded only the following amino acids, identified by chromatography alanine, cysteine, allohydroxyproline, and hydroxy-tryptophan. An investigation of the various peptides obtained on partial hydrolysis led Sorm and Keil to propose for phalloidin, considered to be a... 

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). 

Fixation without Methanol (Hand Devitellinization of Embryos). Often, the highest quality preparations are obtained by not exposing the embryos to methanol, which destroys membrane and other structures. In addition, some reagents such as fluores-cently labeled phalloidin will not work if the embryos have been exposed to methanol. Foregoing the methanol, however, necessitates removing the vitelline membrane by hand. We find this procedure to be most satisfactory (see Protocol 9.3, Method 5). For examples of embryos prepared by this method, see Figure 9.4B,C. 

Figure 9.4. Confocal micrographs of wild-type Drosophila embryos. (A) Whole-mount Drosophila embryos stained with propidium iodide to visuahze the nuclear division cycle. Shown are syncytial embryos in nuclear division cycles 1-13 and interphase of nuclear cycle 14 (cellularization). Bar, 100 pm. (B) Surface views of Drosophila embryos prepared by hand devitellinization and double-stained for actin (fluorescein phalloidin, green) and DNA (propidium iodide, red). Interphase actin caps (top panel) and actin-based metaphase furrows middle panel) are shown for embryos in nuclear division cycle 13. Bottom panel) Embryo at cellularization. Actin-based cellularization furrows surround each of the nuclei. Bar, 10 pm. (C) Surface view of a Drosophila embryo in late metaphase/early anaphase of nuclear division cycle 13. The embryo was prepared by hand devitellinization and triple-stained for actin (fluorescein phaUoidin,green), DNA (propidium iodide,pi/rp/e), and the furrow component, Dah (anti-Dah, Cy5-labeled secondary, red). Both actin and Dah locaUze to the furrows and appear to colocalize in some regions yellow staining, inset). Bar, 10 pm. Inset) 2X magnification.

Sigma carries phalloidin conjugated to FITC, TRITC, and CPITC. It is shipped in rubber-stopper-sealed vials containing 0.1 mg. Inject 4 ml of absolute ethanol into a 0.1-mg vial of phalloidin to prepare a 20 pM stock. 

Prepare 1 pM fluorescently conjugated phalloidin by mixing together the following ... 

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