Technical Reference #344

344.

Phalloidin–Eosin Followed by Photo-oxidation: A Novel Method for Localizing F-Actin at the Light and Electron Microscopic Levels Francisco Capani; Thomas J. Deerinck; Mark H. Ellisman; Eric Bushong; Marketta Bobik; and Maryann E. Martone, University of California San Diego, The Journal of Histochemistry & Cytochemistry, 49(344), (2001)
Link To Paper

Abstract:
We describe a novel high-resolution method to detect F-actin at the light andelectron microscopic levels through the use of the actin-binding protein phalloidin conjugatedto the fluorophore eosin followed by photo-oxidation of diaminobenzidine. This

Keywords:
Cytoskeleton; dendritic spines; electron tomography; 3D reconstruction; purkinje cells

Materials & Methods:
Materials Eosin–phalloidin and rhodamine–phalloidin phalloidin–Alexa- 488 and Alexa568 were obtained from Molecular Probes (Eugene OR). 33 -Diaminobenzidine tetrahydrochloride (DAB) coldwater fish gelatin and the anti- actin antibody were purchased from Sigma (St Louis MO). Paraformaldehyde EM grade glutaraldehyde sodium cacodylate and Durcupan ACM resin were obtained from Electron Microscopy Sciences (Ft Washington PA). Special glass bottomwelled tissue culture plates were obtained from MatTek (Ashland MA). Cell-Tak adhesive was obtained from Collaborative Research (Bedford MA). Tissue We used seven adult male Sprague–Dawley rats in this study. Briefly intracardiac perfusion was performed under deep anesthesia (containing 50 mg/kg ketamine 1 mg/kg rhompun and 5 mg/kg acetopromazine in sterile saline) with normal rat Ringer’s at 35C followed by fixative. For light microscopic analyses rats were perfused with 4% formaldehyde (made fresh from paraformaldehyde) in cacodylate buffer pH 7.2. For -antibody studies the animals were perfused with 4% paraformaldehyde 0.1% glutaraldehyde. The brains were removed and fixed for 2 additional hours in the same solution at 4C. For electron microscopic studies a range of fixative strengths was evaluated containing either 2% or 4% formaldehyde or 0.5%–2.5% glutaraldehyde. The tissue was postfixed for 2 hr in the same fixative. After removal of the brain from the skull coronal or sagittal sections through neostriatum cerebellum and hippocampus were cut at a thickness of 50–80 m on a Leica Vibratome model VT 1000E. Cultured Cells As a control we also labeled cultured bovine aortic endothelial cells (BAECs) fixed using the same conditions as above. These cells possess characteristic bundles of actin filaments called stress fibers. Details about culturing methods are given in Deerinck et al. (1994). Staining Sections with Phalloidin Vibratome sections were washed with 50 mM glycine–PBS containing 0.5% coldwater fish gelatin to block nonspecific binding. After 30 min of washing sections were incubated with agitation in a solution of 4 U eosin–phalloidin and 0.5% coldwater fish gelatin–50 mM glycine–PBS for 2 hr. For light microscopic studies phalloidin conjugated to rhodamine was also used because of its superior fluorescent quantum yield. As a negative control the eosin–phalloidin was omitted. Immunolabeling After repeated rinsing in 0.1 M PBS sections were blocked for 30 min in PBS containing 1% normal donkey serum 1% bovine serum albumin 1% coldwater fish gelatin and 0.2% Triton X-100. Sections were placed in the primary antibody against -actin (dilution 1:100) in 0.1 strength blocking buffer (working buffer) and incubated on a rotator overnight at 4C. After several washes in working buffer a secondary antibody conjugated to FITC was applied for 1 hr at room temperature. For electron microscopic studies an immunoperoxidase procedure was employed. After incubation in the primary antibody the tissue was washed in PBS and incubated for 1 hr in biotinylated anti-mouse IgG (1:200; Vector Laboratories Burlingame CA). The tissue was washed and then incubated for 1 hr in the avidin–biotin– horseradish peroxidase complex (Vector) according to the manufacturer’s instructions. The antigen was visualized by reaction with DAB postfixed with 1% osmium tetroxide for 20 min dehydrated in ascending alcohols and flat-embedded in Durcupan (Fluka; Milwaukee WI). Ultrathin sections were observed on a JEOL 100CX electron microscope. Procedures for immunolabeling cultured cells for tubulin using secondary antibodies conjugated to eosin followed by photo-oxidation can be found in Deerinck et al. (1994). Confocal Microscopy Fluorescent microscopy was performed on a Zeiss Axiovert 35 M inverted light microscope using either a 63 1.4 NA or 40 1.3 NA objective lens. Fluorescent and transmitted light images were recorded using a laser scanning confocal attachment (MRC-1024; Bio-Rad Laboratories Cambridge MA) and a krypton–argon laser.

Microscopic Technique
Electron Microscopy, Light Microscopy

Cell Type(s)
BAEC