Journal of Histochemistry and Cytochemistry, Vol. 45, 595-598, Copyright © 1997 by The Histochemical Society, Inc.


TECHNICAL NOTE

Metal Sandwich Method to Quick-freeze Monolayer Cultured Cells for Freeze-fracture

Toyoshi Fujimotoa and Kazushi Fujimotob
a Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Japan
b Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto, Japan

Correspondence to: Toyoshi Fujimoto, Dept. of Anatomy and Cell Biology, Gunma Univ. School of Medicine, 3-39-22 Showa-machi, Maebashi 371, Japan.


  Summary
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Summary
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Materials and Methods
Results and Discussion
Literature Cited

We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation. (J Histochem Cytochem 45:595-598, 1997)

Key Words: quick freezing, freeze-fracture, freeze replica, plasma membrane, cultured cells, caveolin, immunocytochemistry


  Introduction
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Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

Quick-freezing is considered as the best way to preserve cellular ultrastructure for electron microscopy. Since the advent of the "slammer" (Heuser et al. 1979 ), various instruments based on a similar principle have been developed. However, these instruments are relatively expensive and are not available in every laboratory. Liquid helium, which is recommended for most efficient freezing, is also costly.

Even with a quick-freezing instrument at hand, it is not easy to obtain a wide area of the freeze-fractured plasma membrane, especially from a monolayer of cultured cells. Because the thickness of a monolayer is usually less than 10 µm, a precise knife advance mechanism is required for freeze-fracturing. Unfortunately, by knife fracturing the fracture plane tends to go through the cytoplasm rather than only the cell surface (Neuman 1995 ). These points have constituted practical obstacles to analysis of the molecular architecture of the plasma membrane by freeze-fracture techniques.

We describe here a simple method to quickly freeze monolayer cultured cells by liquid nitrogen. The method does not require any expensive equipment and, more importantly, produces a large area of the fractured plasma membrane each time.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

Cells were grown on gold foil (approximate thickness 20-40 µm) cut into a small trapezoid (approximate size: upper side 2 mm; lower side 4 mm; height 6 mm). The trapezoid was intentionally made asymmetrical so that the cell side could be identified easily in subsequent procedures. Gold foil was kept in 1 N HCl and then in ethanol for more than 30 min each, rinsed with distilled water, and autoclaved.

Various cultured cells were dispersed by trypsin-EDTA, seeded on gold foil in plastic dishes (Figure 1A), and maintained in Dulbecco's modified Eagle's medium with the addition of 10% fetal calf serum. As far as we could determine, gold foil is a good substrate for any cell type without any coating. Because cells on foil cannot be observed by light microscopy, the cell density was inferred from surrounding areas in the plastic dish.



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Figure 1. Schematic drawing of the metal sandwich method. (A) Cells were grown on thin gold foil. (B) Cells on gold foil were inverted on a minimal amount of gelatin on thin copper foil, and the entire sandwich was quickly frozen by pressing between two cooled copper blocks. (C) Cells were fractured by lifting the gold foil. The left and right cells were fractured through the lower and upper plasma membrane, respectively. (D) The fractured surface was replicated by platinum/carbon shadowing: In this case, the P-face of the lower plasma membrane (left) and the E-face of the upper membrane (right) can be observed.

For quick-freezing, a sheet of copper foil (thickness 20 µm) of rectangular shape (approximate size 12 x 20-mm) was prepared. A small amount (5-10 µl) of prewarmed 10% gelatin in PBS was smeared on the copper foil and a piece of gold foil was inverted on the gelatin with the cell side down (Figure 1B). While a corner of copper foil was held with forceps, the sandwich of gold-cell-copper was rapidly pressed between two gold-plated copper blocks that had been precooled in liquid nitrogen. The gold-plated copper blocks were attached to two arms of fine pliers (QF plier; Dosaka EM, Kyoto, Japan) so that the cell sandwich could be grasped in one quick motion. The frozen cell sandwich was kept in liquid nitrogen before further processing.

For freeze-fracturing, the cell sandwich was attached to a specimen table (BB172160-T; Balzers High Vacuum, Balzers, Liechtenstein) by clamping an edge of copper foil by a plate spring; the gold foil was placed on top of the copper foil. The sandwich was transferred to a cold stage of a Bal-zers BAF401 apparatus and a vacuum of 10-7 Torr was obtained. After the temperature was raised to -110C, a cold knife was slowly inserted between the gold and the copper foil. The knife was then moved upwards so that the gold foil was lifted and the frozen cell specimen was fractured (Figure 1C). Platinum/carbon replicas were made as described by the manufacturer (Figure 1D).

The replicas were floated from the specimen in distilled water and transferred to 2.5% sodium dodecyl sulfate (SDS) solution as described (Fujimoto 1995 ). They were stirred in the solution for 1 hr to overnight, rinsed with five changes of PBS, and processed for immunogold labeling. After pretreatment with 1% bovine serum albumin, replicas were transferred to antibody solutions (30-40 µl) placed on Parafilm (American National Can; Greenwich, CT). Primary and secondary antibodies used in the present paper were mouse monoclonal anti-caveolin antibody (clone Z034; Zymed, So. San Francisco, CA) and 10-nm colloidal gold-conjugated goat anti-mouse IgG antibody (Amersham; Poole, UK), respectively. The labeled replicas were rinsed with distilled water, picked up on formvar-coated grids, and observed with a JEOL 2000EX operated at 80 kV. For comparison, some cells were cultured on glass coverslips, "unroofed" as described previously (Fujimoto 1993 ; Fujimoto et al. 1991 ) and labeled with the anti-caveolin antibody for immunofluorescence microscopy.


  Results and Discussion
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Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

Freezing by clamping has already been applied to tissue pieces (Hagler et al. 1983 ), but the critical point of the present method is that cells were sandwiched between gold foil and copper foil before freezing. The space between the cell's lower surface and the gold foil must be well less than 1 µm, as deduced from the distance between cells and other substrates (Chen and Singer 1982 ). By gently pressing the gold foil onto the copper foil, the distance between the cell's upper surface and the copper foil was minimized. Because metal is more efficient than water in heat conduction, the metal sandwich is suitable to freeze both surfaces of monolayer culture cells efficiently.

Replicas obtained by the method were mostly from the fractured plasma membrane. Fracture through the cytoplasm or the extracellular space seldom occurred. Because of the mounting direction of the cell sandwich, fracture planes observable by the present procedure were either the E-face of the upper (dorsal) membrane or the P-face of the lower (ventral) membrane (Figure 1C). The upper and lower membranes are the plasma membranes facing the copper foil and the gold foil, respectively. The proportion of the upper and lower plasma membranes varied for different cell types and probably for different culture conditions, but both membranes can be observed in every replica.

The replicas of the membrane can be used for immunolabeling after treatment with SDS (Fujimoto et al. 1996 ; Fujimoto 1995 ). For the P-face of the lower plasma membrane to be labeled by antibodies, epitopes must be on the cytoplasmic side. In the E-face of the upper plasma membrane, epitopes on the extracellular surface can be labeled in the replicas. As an example of the P-face of the lower plasma membrane, we examined whether caveolin can be immunolabeled. Caveolin is an integral membrane protein that is presumed to adopt a hairpin loop structure with both N and C terminals exposed to the cytoplasmic surface (Monier et al. 1995 ; Dupree et al. 1993 ). As shown in Figure 2, labeling for caveolin occurred in a specific manner. Caveolae were observed as dimples of various depth or fractured at their orifice. Caveolae seen as dimples or invaginations were invariably decorated with colloidal gold particles. On the other hand, caveolae fractured at the orifice were not labeled.



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Figure 2. Immunogold labeling of caveolin on the P-face of the lower plasma membrane of a cultured human fibroblast. Caveolae are observed as dimples (large and small arrows) or as craters (arrowheads): The latter are caveolae fractured at the orifice. Colloidal gold particles are observed at the bottom of large dimples (large arrows) and around the smaller pits (small arrows), but not around craters (arrowheads). Bar = 200 nm.

Figure 3. Immunofluorescence microscopy of caveolin observed in the lower plasma membrane of a cultured human fibroblast. Labeling for caveolin is observed as a single dot or aggregated dots.Bar = 1 µm.

Because this method provides a large planar view of the plasma membrane, the replica obtained can be directly correlated with the result of immunofluorescence microscopy. Figure 3 shows immunofluorescence labeling of caveolin in the lower plasma membrane preparation: In this preparation, the upper plasma membrane and the cytoplasm were removed before immunolabeling. Caveolae are seen as discrete dots and are densely distributed in some areas. Figure 2 apparently corresponds to a region in which caveolae form a dense patch.

The present method was developed based on the one developed by Pauli et al. 1977 . They described a fracturing method for fixed cultured cells: a highly viscous mixed solution of polyvinyl alcohol and glycerin was placed on a specimen stub, and fixed and glycerinated cells on coverslips were inverted on the solution. Therefore, the orientation of the specimen during fracturing and obtainable fracture faces is the same as in the present method. The polyvinyl alcohol-glycerin mixture provided a good support for freezing but was deleterious to the ultrastructure of unfixed cells. We used a small amount of 10% gelatin in PBS instead. When fractured, the fracture plane almost always went through the upper or the lower plasma membrane and never through the gelatin layer.

With a "slammer" or with instruments based on a similar principle, frozen specimens must be cut by a knife to obtain fracture faces. In thin monolayer culture cells, cutting at an exact height is not necessarily easy. However, by lifting off the gold foil, fracture almost always occurred through the cell membrane. The other merit of the metal sandwich method is that large fracture faces of the plasma membrane are revealed each time. Obviously, this metal sandwich can also be frozen by a more sophisticated metal block freezing apparatus, but the metal foil might scratch the mirror surface on contact.

Now that freeze replicas can be a substrate for immunocytochemical labeling, the present method should be useful in revealing the molecular architecture of the plasma membrane in a variety of cultured cells.


  Acknowledgments

Supported by a grant-in-aid for Scientific Research (B) (no. 08457001) from the Ministry of Education, Science, Sports, and Culture of the Japanese Government and by a research grant from the Ciba-Geigy Foundation (Japan) for the Promotion of Science.

We thank Ms Natsuko Hatanaka, Yukiko Takahashi, and Fujie Miyata for excellent technical and secretarial assistance.

Received for publication September 4, 1996; accepted November 22, 1996.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

Chen WT, Singer SJ (1982) Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol 95:205-222[Abstract]

Dupree P, Parton R, Kurzchalia TV, Simons K (1993) Caveola and sorting in the trans-Golgi-network of epithelial cells. EMBO J 12:1597-1605[Abstract]

Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins--application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108:3443-3450[Abstract/Free Full Text]

Fujimoto T (1993) Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol 120:1147-1157[Abstract]

Fujimoto T, Lee K, Miwa S, Ogawa K (1991) Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro. J Histochem Cytochem 39:1485-1493[Abstract]

Fujimoto K, Umeda M, Fujimoto T (1996) Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling. J Cell Sci 109:2453-2460[Abstract/Free Full Text]

Hagler HK, Lopez LE, Flores JS, Lundswick RJ, Buja LM (1983) Standards for quantitative energy dispersive X-ray microanalysis of biological cryosections: validation and application to studies of myocardium. J Microsc 131:221-234[Medline]

Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, Evans L (1979) Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol 81:275-300[Abstract]

Monier S, Parton RG, Vogel F, Behlke Y, Henske A, Kurzchalia TV (1995) VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro. Mol Biol Cell 6:911-927[Abstract]

Neuman TM (1995) A guide to equipment for production of freeze-fracture replicas. In Severs NJ, Shotton DM, eds. Rapid Freezing, Freeze-Fracture, and Deep Etching. New York, Wiley-Liss, 51-67

Pauli BU, Weinstein RS, Soble LW, Alroy J (1977) Freeze-fracture of monolayer cultures. J Cell Biol 72:763-769[Abstract]