Ethane-Freezing/Methanol-Fixation of Cell Monolayers: A Procedure for Improved Preservation of Structure and Antigenicity for Light and Electron Microscopies

Abstract

In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events, and organelle movements, it is necessary to fix/stop timedependent events in the millisecond range. Ideally, immunoelectron microscopical labeling experiments require the availability of high-affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF) and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton. 1998 Academic Press

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

“…Staining of F-actin was performed with Oregon Green-phalloidin (Molecular Probes, Eugene, OR) for 60 min. The rapid freezing combined with fixation and permeabilization in ultracold methanol was performed as described (Neuhaus et al, 1998). Slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and kept at 4°C in the dark.…”

“…It is reasonable to assume that the tail of TgM-A, being very rich in arginine and lysine residues, aldehyde cross-linking to plasma membrane proteins, may lead to complete epitope masking. Therefore, we used an alternative fixation method involving rapid freezing fixation and permeabilization in ultra-cold methanol, an ideal method to preserve both structure and antigenicity (Neuhaus et al, 1998). Figure 4C illustrates that both the endogenous TgM-A ( Figure 4C, b) and the myc-tagged protein ( Figure 4C, a) were now detected by the anti-tail antibody.…”

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Coronin (_) is an actin-binding protein localised to the cortex and in the actin coat of newly formed macropinosomes, as well as post-Iysosomes before they acquire vacuolin (monoclonal antibody 176-3-6 (de Hostos et al 1993), gift from Dr. G. Gerisch, MPI for Biochemistry, Munich). The vacuolar-H+-ATPase ( ) is associated with membranes of the contractile vacuole complex and, to a much lesser extent with endosomes (monoclonal antibody 221-35-2 against the A-subunit of the vacuolar-H+-ATPase (Neuhaus et al 1998)). The common Antigen-1 ( • ) is a M-6-P-containing epitope shared by D. discoideum lysosomal enzymes such as (-mannosidase and (-glucosidase (Freeze et al 1990) (monoclonal antibody 221-342-5 (Neuhaus et al 1998)).…”

“…The vacuolar-H+-ATPase ( ) is associated with membranes of the contractile vacuole complex and, to a much lesser extent with endosomes (monoclonal antibody 221-35-2 against the A-subunit of the vacuolar-H+-ATPase (Neuhaus et al 1998)). The common Antigen-1 ( • ) is a M-6-P-containing epitope shared by D. discoideum lysosomal enzymes such as (-mannosidase and (-glucosidase (Freeze et al 1990) (monoclonal antibody 221-342-5 (Neuhaus et al 1998)). Dynamin A (_) is a GTPase potentially involved in vesicle scission and is localised around post-lysosomal vacuoles in a punctate fashion (GFP-dynaminA (Wienke at al.…”

Nucleomorphin

“…Additionally, we also observed that fluorescence levels varied broadly from cell to cell, a common problem related to the actin15-promoter used to drive expression of the GFP fusion protein (55,56). In vegetative cells, GFP-NumA was almost exclusively within the nucleus appearing as distinct arc-like bands that corresponded to heterochromatin-like domains adjacent to the nuclear membrane (57) (Fig. 8B).…”