We express our gratitude to M. Bouchecareilh, E. T&#233;taud, S. Duvezin-Caubet, and A. Devin for their help and comments on the manuscript. We are grateful to the electronic imaging pole of Bordeaux Imaging Centre where the images were taken. This work was supported by Centre National de la Recherche Scientifique.

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.
<ol>
	<li>Prepare a 100 mL solution of 0.3% formvar in chloroform. Allow it to dissolve overnight without agitation.</li>
	<li>Put 400 mesh (number of holes per square inch)electron microscopyc...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+living+cells:+principles+and+practice.">Cryopreservation of living cells: principles and practice.</a> Transfusion. 47 (5), 935-945 (2007).</li><li>Vanhecke, D., Graber, W., Studer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close-to-native+ultrastructural+preservation+by+high+pressure+freezing.">Close-to-native ultrastructural preservation by high pressure freezing.</a> Methods Cell Biol. 88, 151-164 (2008).</li><li>Bobik, K., Dunlap, J. R., Burch-Smith, T. 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The authors declare that they have no conflicts of interest.

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...

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 alternative for the preservation of cellular structures3, keeping them intact. The highest quality of TEM images4 in thin resin sections can be obtained using the cryofixation/freeze substitution method. The aim of this technique is to obtain vitrified biological samples without ice crystals formation or containing ice crystals small enough not to damage the ultrastructure of the cells. High-pressure freezing (HPF) and ultra-rapid cryofixation, which is also called plunge freezing (PF), are two methods to cryofix samples. HPF immobilizes molecules in the cell instantaneously and avoids the damage caused by conventional EM fixation. Several types of freezing machines and automatic substitution devices have been developed5. The freezing machines and consumables (liquid nitrogen, specimen carriers, etc.) are expensive, but they allow for producing high-quality electron micrographs6,7. PF is a technique that was used in the early 1950s and is described in the literature as simple and cheap8.During the PF procedure, the prepared sample is frozen at a rapid rate to obtain ice crystals size less than 3 to 5 nm. To this end, the sample is plunged into a liquid cryogen, such as ethane, propane, or an ethane-propane mixture. Since the 1950s, improvements of PF have been done to make this technique available to a greater number of users. High-pressure freezing is currently the only viable way to freeze a large variety of samples thicker than 50 &#181;m (up to a thickness of 200 &#181;m for disc-shaped samples)5, whereas PF is widely used to image small objects (&lt;100 nm), such as macromolecular complexes suspended in a thin film of amorphous ice9. Larger samples, for example eukaryotic cells, can be cryofixed by PF, but requires specimen holders, such as capillary copper tubes or sandwich systems8,10,11. 
Here, a rapid, easy-to-use and low-cost plunge freezing/freeze-substitution technology usable for various suspension cell cultures is presented.

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plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

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.

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

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

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

The goal of the following experiments is to propose a tool for ultrastructural and immunolocalization studies of suspension cells for visualization in transmission electron microscopy. This technique is easy to use, low cost, and gives excellent preservation of cellular structure. This is a culture of suspension cells.First, the cells are harvested by different centrifugations to obtain a pellet in a microcentrifuge tube. In a second step, the sample is rapidly frozen by plunge freezing which immobilizes the cells and limits the formation of ice crystals. Next, freeze substitution is performed in order to replace water by solvent containing osmium tetroxide as fixative to stabilize cell structures.Results show that plunge freezing, and freeze substitution, preserve perfectly the infrastructure of the cells. Chemical fixation was a primary method for fixing tissue or cells for transmission electron microscopy, but with this method, artifacts and extraction of cellular contents are usually observed. An alternative to chemical fixation is cryofixation.This method represents the best choice for producing the highest quality of morphology in thin resin sections. Plunge freezing is a cryofixation technique that immobilizes molecules in the cell instantaneously to avoid damage caused by conventional chemical fixation. Before plunge freezing the sample, grid cutted with Formvar film must be prepared to be used as support for freezing.Polish glass slide with lint-free cloth until shiny and wipe clean. Dip the glass slide in a solution of Formvar 0.3%in chloroform a few seconds and leave to dry upright in a beaker. Manipulation must be realized under a fume hood using personal protective equipment due to the presence of chloroform.After filling a crystallizer with distillated water, pass over the water surface a glass bar to remove dust. When the film is dry, score the edges with a sharp razor blade so that the film may lift off. Frost the slide by exhaling heavily onto the slide to get a layer of water vapor on the film.Immediately float the film onto the water surface by touching the bottom of the slide with an angle about 30 degrees to the water surface. Slowly lower the slide into the bath, teasing the film off as necessary. The Formvar film settles slowly on the surface of the water.Attach the film on the edge of the crystallizer using tweezers. Gently lay the 400 mesh copper grids face-down on the film in the regions that are of the proper thickness and without wrinkles. The film with grids is picked up by covering them with Joseph paper while they float on the water surface.Pick up the edge of the Joseph paper and the film with grids should come with it. Don't let your fingers touch the water surface or you run the risk of leaving a film in the water. Let the grids dry several hours before using them.For ultrastructural studies, put osmium tetroxide ampoule in a glass flask. Shake well the flask to break the ampoule. Add appropriate volume of acetone 100%to have a final concentration of 4%Stir the flask containing the freeze substitution medium, and dispense 1.5 milliliter aliquots into cryovials with plastic transfer pipet.Close all caps and keep in freezer at minus 84 degrees before using. For protein immunolabeling studies, prepare 0.1%of uranyl acetate in acetone. When the solution becomes clear, filtrate using 0.2 micrometer filter and dispense 1.5 milliliter aliquots into cryovials.Keep in freezer at minus 84 degrees before using. To be sure to have the appropriate consistency of pellets from different cell types, raw yeast cells in minimum of complete liquid medium to obtain one 10 to the nine cells. Escherichia coli in DTY medium to obtain five 10 to the 10 bacterial cells.Leishmania flagellum in AM medium and Trypanosoma brucei in SDM79 medium to obtain five 10 to the eight parasite cells. Transfer the culture medium containing suspension cells to a 50 milliliter propylene tube and centrifuge for three minutes at 1, 500 g. Remove the supernatant and resuspend the pellet in one milliliter of respective cell culture medium.Centrifuge in a microcentrifuge tube for one minute at 3, 900 g. Remove the supernatant completely. For plunge freezing method, propane bottle with a tube connected to the propane cylinder must be used.Propane being potentially explosive, realize the liquefaction in a well-ventilated room or in a fume hood. No open flames are allowed. Personal protective equipment including cryogloves must always be worn when using liquid nitrogen.Put a little quantity of liquid nitrogen in polystyrene tray. Place the brass cup in liquid nitrogen. Bubbles appear.Wait until the bubbles subside. The brass cup is then frozen. Put the tube connected to the propane cylinder in contact with the brass cap wall.Gently open the propane cylinder valve. A lot of smoke forms as propane does not begin to liquefy. When the propane begins to liquefy, the smoke disappears.Stop the liquefaction by closing the propane-cylinder valve and removing rapidly the tube connected to the propane cylinder. Fill the polystyrene tray with liquid nitrogen up to five millimeters from the top of the brass cup. Freeze little brass cups where we will replaced the sample after freezing by putting them in liquid nitrogen.Put one cup for one sample. Be careful not to mix samples. To obtain the appropriate consistency of the pellet, it just necessary to respect culture conditions and centrifugation parameters indicated previously.Freeze the tweezers before using them. Take a 400 mesh copper microscopy grid cutted with Formvar. Plunge a double-edge dissecting needle in the pellet.Put, using the tool, a drop of pellet on the grid. Very rapidly plunge the grid into liquid propane and stir a few seconds. Rapidly transfer the frozen grid in the little brass cup.For each sample, make at least three grids. Don't forget to freeze the tweezers before taking up a grid. Freeze the tweezers before using them.Rapidly remove the cup and transfer the frozen grids into the cryovial using the tweezers. Put the cap back on the cryovial. Repeat the operation for each grid of each sample.Close caps and stir the cryovial to be sure the samples are in freeze substitution medium. Let the cryovials for three days in the minus 84 degrees freezer. Carry the cryovials in a polystyrene tray to avoid a sudden rise in temperature to the minus 30 degree freezer.Let one hour at minus 30 degrees. Carry the cryovials at minus 15 degrees for two hours, then put the samples at four degrees for two hours, and 30 minutes at room temperature. Remove the freeze substitution medium using plastic transfer pipettes and put it in an appropriate waste bottle clearly identified.Put about 500 microliters of 100%acetone. Open the glass vial and stir the cryovial and rapidly put the freeze substitution medium in a glass vial. Rinse one cryovial with 100%acetone.Rinse three times 10 minutes with 100%acetone. Carry the cryovials in a polystyrene tray to avoid a sudden rise in temperature to the minus 30 degree freezer. Let two hours at minus 30 degrees.After putting samples in a polypropylene vial, replace the freeze substitution medium by fresh freeze substitution medium and let two hours at minus 30 degrees. Rinse one hour with 100%acetone and three times one hour with 100%ethanol. Plunge freezing/freeze substitution method resulted in excellent preservation of the intracellular structures of suspension cells.In Saccharomyces cerevisiae, the structures are not shrunken and the cytoplasm appears dense. Moreover, membranes are smooth, which is a proof of a good preservation. All organelles, such as nucleus, mitochondria, and vacuoles can be perfectly identified.Excellent preservation is also observed in Leishmania flagellum, Trypanosoma brucei, and Escherichia coli. In nucleus, double membrane forming nuclear envelope, nuclear pore, spindle pole body, and ribosomes attached on the outer membrane of the nuclear envelope are distinctly visible. In mitochondria, the internal membrane invaginations, called cristae, are clearly visible.Critical organelles are the vacuoles, because large ice crystals can develop easily. In the image, vacuoles are perfectly preserved without ice crystal damage. Plunge freezing/freeze substitution method can be also used to perform protein immunolocalization.The procedure preserves perfectly protein antigenicity, and as mentioned previously, organelles are clearly identified. Different antibodies have been already tested to target different proteins in yeast, such as anti-ATP synthase and anti-porine. Such experiment is also possible for different experimental systems, as in Leishmania, where the flabarin protein was localized.After watching this video, you should have good understanding how to prepare samples with plunge freezing/freeze substitution method for visualizing them in transmission electron microscopy. This technique is rapid, easy to use, low cost, and give excellent preservation of cell infrastructures. Be careful with liquid nitrogen, but especially with osmium tetroxide and uranyl acetate.They should be handled in the fume hood while wearing appropriate personal protective equipment, including lab coat and gloves.

plunge-freezing-tool-for-ultrastructural-immunolocalization-studies

Research

JoVE Journal

Biology

10.7K Görüntüleme.  Université de Bordeaux. The goal of the following experiments is to propose a tool for ultrastructural and immunolocalization studies of suspension cells for visualization in transmission electron microscopy. This technique is easy to use, low cost, and gives excellent preservation of cellular structure. This is a culture of suspension cells.First, the cells are harvested by different centrifugations to obtain a pellet in a microcentrifuge tube. In a second step, the sample is rapidly frozen by plunge freezin...