Improved visualization of F-actin in the green alga Acetabularia by microwave-accelerated fixation and simultaneous FITC-Phalloidin staining.
By employing a new procedure we have been able to visualize a highly intense actin cytoskeleton in the unicellular green alga Acetabularia acetabulum Silva. The protocol described in this study involves microwave-accelerated simultaneous permeabilization with 10% dimethyl sulphoxide, fixation with 1% glutaraldehyde and incubation with 0.5 microM fluorescein-isothiocyanate-conjugated Phalloidin. Comparison of the images of the actin cytoskeleton of the stalk, as visualized by methods used previously, with those obtained in our own experiments shows that the actin filaments were preserved completely in an excellent condition. The required time for each procedure could be reduced from 12 h for the most commonly used immunofluorescence technique to 35 min. Moreover, it has been possible to observe the actin filament system of hair whorls, rhizoid and tip. Previously, the actin cytoskeleton of these parts of the cell could not be visualized by conventional techniques. It is shown that each region of the cell-stalk, tip, rhizoid and side branches-displays characteristic degrees of actin bundling and regularity of actin alignment.
Sawitzky
H
H
Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg, Germany.
Willingale-Theune
J
J
Menzel
D
D
D000199:Actins / Q000648:ultrastructure*; D000460:Chlorophyta / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D005452:Fluoresceins / Q000737:chemistry*; D008872:Microwaves; D010590:Phalloidine / Q000031:analogs & derivatives* / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
D000199:Actins; D005452:Fluoresceins; C037191:fluorescein isothiocyanate-phalloidin; D010590:Phalloidine