Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Summary

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

Abstract

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

Introduction

Sample preparation is critical for the success of any electron microscopy study. Conventional EM fixation was the primary method for fixing tissue or cells for transmission electron microscopy (TEM)¹. First, aldehydes and osmium tetroxide are used to chemically fix the material at room temperature. Then, the material is dehydrated with organic solvents, infiltrated, and embedded in epoxy resin. This method is dependent upon the penetration rate of fixatives into the cell. Consequently, the artifacts and extraction of cellular contents are usually observed².

Cryofixation is clearly a better alt....

Protocol

1. Preparation of Formvar Grid Film

NOTE: Perform chloroform manipulation under a fume hood using personal protective equipment (gloves, lab coat, glasses). Use 400 mesh electron microscopy copper grids. With other grid types (other mesh sizes, gold and nickel grids), the quality of freezing is worse.

1. Prepare a 100 mL solution of 0.3% formvar in chloroform. Allow it to dissolve overnight without agitation.
2. Put 400 mesh (number of holes per square inch)electron microscopycopper grids (diameter 3.05 mm) in a glass vial (height 3 cm and diameter 2 cm) containing acetone. Stir the glass vial with a circular movement of the....

Results

In this article, an easy-to-use and low-cost plunge freezing method for ultrastructural (Figures 1 and Figure 2) and immunolabeling (Figure 3) studies is presented. We demonstrate that it is not necessary to have special equipment for the freeze-substitution procedure and that the warming procedure takes less than 6 h instead of the 24 h using a dedicated system.

Discussion

TEM is a powerful method for the ultrastructural observation of organelles, cells, and tissues. Cryofixation/freeze-substitution is currently the best method for the preservation of both ultrastructure and protein antigenicity. Chemical fixatives penetrate and act very slowly thereby allowing structural rearrangements before the complete stabilization of the ultrastructure². Conversely, cryofixation/freeze-substitution instantly stabilizes cellular structures⁴. However, cry.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Grids	Electron Microscopy Sciences	T400-Cu
Formvar	Electron Microscopy Sciences	15800
Propane N35
Liquid nitrogen
Double edge dissecting needle	Electron Microscopy Sciences	72947
Cryovials	Electron Microscopy Sciences	61802-02
Osmium tetroxide	Electron Microscopy Sciences	19130
Glass vial 8.5 mL	Electron Microscopy Sciences	64252
Uranyl acetate	Electron Microscopy Sciences	48851
Acetone	Sigma	32201
Ethanol 100%	Sigma	32221
Glass slides	VWR international	631-9439
Tweezers
Acrylic resin	Electron Microscopy Sciences	104371
Epoxy resin M	Sigma	10951
Epoxy resin M hardener	Sigma	10953
Dibutyl phtalate	Sigma	80102
Epoxy resin M accelerateur	Sigma	10952
Crystallizer	Fischer scientific	08-762-9
Joseph paper	VWR international	111-5009
Brass cups			do it yourself shop or made by yourself
Saccharomyces cerevisiae
Shizosacharomyces cerevisiae
Trypanosoma brucei
Leishmania amazonensis
Escherichia coli
Airtight box	Fischer scientific	7135-0001
Air purifying respirator	Fischer scientific	3M 7502
Cartridge for respirator	Fischer scientific	3M 6001
Particulate filter	Fischer scientific	3M 5N11