ARTICLE |
Correspondence to: Judith Klumperman, Dept. of Cell Biology, AZU Rm G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: J.Klumperman@lab.azu.nl
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Summary |
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Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative lightelectron microscopy.
(J Histochem Cytochem 50:10671080, 2002)
Key Words: cryoultramicrotomy, immunogold, PC12 cells, hippocampal neurons, electron microscopy, flat-embedding
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Introduction |
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COLLOIDAL GOLD is a powerful tool for the detection of immunolabeled molecules at the ultrastructural level (
The immunogold labeling technique can be combined with various preparation techniques. In pre-embedding protocols, cell membranes are made permeable to the immunoreagents by chemical or mechanical treatment. These approaches usually result in high labeling densities, but permeabilization may lead to a severe loss of fine structural integrity. Permeabilization is avoided in postembedding techniques, in which immunoreactions are performed on the surface of ultrathin sections of plastic- or Lowicryl-embedded cells or tissues. A drawback of postembedding methods, however, is that the antigenicity of molecules is often severely affected by the harsh treatment with organic solvents and dehydration of the specimen necessary for embedding.
The disadvantages of the pre- and postembedding techniques are avoided by ultracryotomy, a method by which ultrathin sections are prepared from non-permeabilized, non-embedded mildly fixed cells or tissue (
The preparation of cryosections of cultured cells involves a mild fixation, after which cells are removed from the carrier substrate with a cell scraper or enzymatic proteolysis and subjected to pelleting (
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Materials and Methods |
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Antibodies
Rabbit antisera against vesicle-associated membrane protein-2 (VAMP-2) and synaptophysin, and mouse monoclonal antibody against rab3 A/B/C/D (Cl 42.1;
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Culture of PC12 Cells
PC12 cells (clone 251-II) were routinely grown on 35-mm petri dishes in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal calf serum at 37C under 10% CO2 (
Low-density Cultures of Hippocampal Neurons from Mouse Brain
When grown at low density, hippocampal neurons go through five clearly defined developmental stages that closely resemble the in vivo situation (
Immunofluorescence
Hippocampal neurons grown for 14 days on coverslips were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4C. Fixative was removed by three washes in PBS. After quenching free aldehyde groups with 50 mM NH4Cl in PBS for 5 min and an additional wash in PBS, the cells were permeabilized for 1 hr in 0.5% BSA in PBS containing 0.1% saponin (blocking solution). The BSA in this solution reduces nonspecific proteinprotein interactions of the antibodies. Cells were incubated with an MAb against VAMP-2 for 30 min and then washed three times for 5 min with blocking solution. A Cy3- (Jackson Laboratories; West Grove, PA) or FITC- (DAKO; Glostrup, Denmark) conjugated rabbit anti-mouse IgG secondary antibody was diluted in blocking solution and applied for the next 30 min. After several washes, cells were mounted in Mowiol on an object slide and viewed with a x63 planapo objective on a Leitz DMIRB fluorescence microscope (Leica; Voorburg, The Netherlands) interfaced with a Leica TCS4D confocal laser scanning microscope (Fig 7), or on a Leica Polyvar epifluorescence microscope (Fig 8A).
Standard Procedure for Preparation of Ultrathin Cryosections from Cultured Cells
Cryosections from cells grown on a solid substrate can be prepared using various adaptations of the standard protocol developed by
In general, cells are fixed at a minimum for 2 hr at room temperature (RT) or overnight in the cold. The fixative is rinsed away by washing twice with PBS to which 0.02 M glycine is added to quench free aldehyde groups. A small volume of 1% gelatin (Twee Torens; Delft, The Netherlands) in PBS at RT is then added, in which the cells are removed from the dish with a cell scraper. Cells are transferred to an Eppendorf vial and centrifuged for 3060 sec at 1500 rpm, after which the 1% gelatin is replaced by a 12% gelatin solution at 37C. After resuspension, cells are either centrifuged again, after which the excess gelatin is removed (procedure A), or transferred to a microscope slide (procedure B). In the latter case, a small petri dish is placed, bottom down, on the top of the droplet to transform it into a slab. In both procedures the gelatin solution is solidified for 30 min on ice. The 12% gelatin provides the necessary support to handle the cells during the following preparation steps, which are all performed in the cold room.
In procedure A, the bottom of the Eppendorf tube is cut off with a razor blade, after which 1-mm3 gelatin blocks are prepared. Similar blocks can be directly prepared in procedure B. The small blocks are rotated in 2.3 M sucrose in phosphate buffer for at least 4 hr at 4C. Sucrose acts as a cryoprotectant, which abolishes ice crystal formation during the subsequent freezing step. After sucrose impregnation the blocks are placed on top of an aluminum specimen holder, the excess of sucrose is removed, and the specimen plus holder are quickly plunged into liquid nitrogen.
Sectioning begins with removal of the sucrose cover and flattening of the front of the specimen. The sides of the specimen are trimmed with a razor blade, the corners of a glass knife, or with a specialized trimming diamond. After trimming the specimen to an optimal rectangular shape, ultrathin sectioning with a diamond knife is performed at about -120C. Sections of 6065-nm thicknesses are removed from the knife with a 1:1 mixture of 2% methylcellulose and 2.3 M sucrose (
This procedure results in the random visualization of cell profiles of pelleted cells. Examples of data obtained according to this procedure are numerous. For recent studies see, e.g.,
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Results |
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Preparation of a Cell Slab from a Plastic Petri Dish
To overcome the necessity to make cell pellets and be able to section cells in parallel to the dish, we have developed a method that avoids scraping of the cells and allows removal of intact cells from a plastic petri dish (method A in Fig 1). We performed our initial studies with non-differentiated PC12 cells fixed after 7 days of culture. Non-differentiated PC12 cells are grown directly on the petri dish, but experiments with poly-L-lysine- and collagen-coated petri dishes were equally successful. Cells were fixed in a mixture of 2% formaldehyde (freshly prepared from paraformaldehyde) and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After quenching with 0.02 M glycine, an extra wash step with 0.1% BSA in PBS was performed, after which the fixed cell monolayer was covered with a thin layer of 12% gelatin in PBS pre-warmed to 37C. We used 750 µl 12% gelatin for a 35-mm and 2 ml for a 60-mm petri dish. We found that the added wash with BSA considerably facilitated spreading of the 12% gelatin solution. The gelatin was solidified at RT and rinsed once with 0.1% BSA in PBS. Then a second thin layer of 12% gelatin containing fixed erythrocytes was added on top of the first layer and solidified at 4C (Fig 1A1). To prepare the gelatin/erythrocyte slab, freshly isolated erythrocytes were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 2 hr and pelleted by a 3060-sec centrifugation in an Eppendorf centrifuge. Subsequently, 100 µl of the erythrocyte pellet was dissolved in 5 ml of 12% gelatin solution. This second layer facilitated orientation of the block later on and provided the necessary thickness for sectioning.
To enhance sucrose infiltration in the double-gelatin layer, the gelatin was scarified from top to bottom with a scalpel (Fig 1A2). Cells were then subjected to a prolonged infiltration with 2.3 M sucrose for 4872 hr at 4C on a rocker. The sucrose infiltration, in addition to cryoprotection, caused a slight shrinking of the gelatin layers, resulting in loosening of the gelatin slab plus cells from the dish. Notably, because the gelatin melts when a section is transferred to RT, at which the labeling procedure is carried out, it does not interfere with the immunoreaction. Large pieces of the gelatin/cell slab could then be detached from the dish with a thin spatula (Fig 1A3). These blocks were stained for 30 min at 4C in a solution of 1% Toluidine blue to which 2.3 M sucrose was added to better preserve ultrastructure. The Toluidine blue staining is optional. We found that it allowed detection of cells in 1-µm cryosections that were viewed under the binocular and therefore enabled selection of areas of interest for further processing. Moreover, it facilitated ultrathin sectioning because the stained cells were clearly visible in the microtome. Finally, the Toluidine blue staining was used as a control to check whether cells had accidently remained attached to the petri dish during the flat-embedding procedure. After selecting specific areas under the binocular, approximately 1-mm3 blocks of the gelatin slab were cut out with a razor blade, mounted on aluminum pins (Fig 1C1), and frozen in liquid nitrogen. For sectioning in parallel to the dish, the erythrocyte layer was oriented towards the pin (Fig 1C1a). Alternatively, the erythrocyte layer can be placed sideways (Fig 1C1b), which is of use when, for example, polarized epithelia are sectioned. A light microscopic image of a 1-µm-thick cryosection, prepared in the orientation as shown in Fig 1C1b, is shown in Fig 2. For our goal, ultrathin cryosections were cut parallel to the monolayer, picked up in a 1:1 mixture of 2.3 M sucrose and 2% methylcellulose (
The flat-embedding procedure resulted in a superb ultrastructural preservation of non-differentiated PC12 cells (Fig 3 and Fig 4). Staining with Toluidine blue (optional) resulted in a denser appearance of the cells in the EM, especially the nucleus (Fig 3A and Fig 3B) but had no effect on labeling densities (Fig 3C3F). Although designed for polarized cells, the flat-embedding approach also proved to be of interest to view non-differentiated cells. As shown in Fig 4, in areas of close contact PC12 cells alternate large areas of flattened plasma membrane with regions displaying microvilli. This type of ultrastructural information is only poorly or incompletely visualized when cells are scraped from the dish.
Preparation of a Cell Slab from a Glass Coverslip
Primary cultures of hippocampal neurons are an often used model system to study transport and development of polarized neuronal cells (
Because the neuronal extensions are directly opposed to the cutting edge of the knife (Fig 1C2) and have a limited height, they are present in the first 2030 sections only. Therefore, blocks were not trimmed at the front. Ultrathin cryosections of hippocampal neurons embedded according to this procedure revealed a network of dendritic and axonal extensions emerging from the cell body (Fig 5). Immunogold labeling clearly provided important information to understanding of the complex architecture and the distinct membrane domains of the neuron. For example, immunogold labeling for rab3 revealed that some of the membrane-bounded structures close to the cell body contained high levels of rab3-positive synaptic vesicles (see Fig 5 inset), suggesting that these are cross-sections of synapses contacting the cell body. Immunogold labeling also discriminated axonal from dendritic extensions (Fig 6A). Axons but not dendrites contained high levels of rab3 (not shown) and VAMP-2 (Fig 6A). Many contact sites between axons and dendrites were observed, sometimes with the synaptic specializations clearly visible (Fig 6C). Synapses had a bulb-like (Fig 6B) or elongated (Fig 6C) shape.
CLEM
An important additional advantage of the flat-embedding technique is the possibility of performing CLEM. Staining of hippocampal neurons with VAMP2 by immunofluorescence results in a patchy staining pattern (Fig 7). Often two fluorescent tracks are running in parallel, delineating an unstained area (see also Fig 7 inset). With the information obtained in the EM (Fig 6), this peculiar staining pattern could be explained as a multitude of bulb-like and flattened synaptic contact sites outlining the postsynaptic dendrite. Although informative, this type of CLEM is indirect. For direct CLEM we subjected fluorescence-stained cells to the flat-embedding procedure. First, cells were stained for VAMP-2 by the routine fluorescence procedure described in the methods section, using an FITC-conjugated secondary antibody (Fig 8A). After photographing areas of interest in the fluorescence microscope, the coverslip with cells was removed from the object slide by rinsing in PBS, after which the procedure was carried out as described above. Areas selected under the fluorescence microscope were backtracked under the binocular, which was feasible because of the Toluidine blue staining. Cryosections of the selected areas were then made using the flat-embedding procedure and sections were labeled with anti-FITC antibody and 10-nm gold. Fig 8 shows a sequence of pictures of increasing magnification, illustrating how a fluorescent spot of VAMP2 (arrow in Fig 8A) is identified as a synaptic terminal in the electron microscope (arrows in Fig 8B8D). At high magnification, it becomes clear that the gold particles indicating the presence of VAMP-2 are associated with the small synaptic vesicles in the synaptic terminal (Fig 8E). Equivalent data were obtained when an Alexa 488-conjugated secondary antibody was used for immunofluorescence and an anti-Alexa antibody on the ultrathin cryosections (not shown). This CLEM procedure resulted in good overall labeling density but the overall morphology at the ultrastructural level decreased compared to flat-embedded cryosections not previously stained for immunofluorescence, probably because of the saponin permeabilization step in the fluorescence procedure. Permeabilization can be avoided when proteins are made fluorescent by recombinant tagging with GFP or its derivatives and using an anti-GFP antibody for labeling of the cryosections. Finally, when serial sections are collected, multiple cross-sections of the structures previously identified by immunofluorescence can be examined, yielding possible additional information (not shown).
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Discussion |
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The in situ localization of molecules at the subcellular level by immuno-EM has proved to be an important method in basic and applied cell biology. In light of the increasing demand for localization information on newly discovered proteins, advances in technology and improvement of existing methods are highly desirable. Previously, we reported that the retrieval of cryosections with a mixture of sucrose/methylcellulose results in superb ultrastructural detail, which is especially of value for lipid-rich regions in the cell, such as the endoplasmic reticulum to the Golgi intermediate compartment (
The technical difficulty we had to overcome was to remove cells from the substrate without disturbing their morphological integrity. We found that embedding of cells in a slab of 12% gelatin provided the necessary support to remove cells undamaged.
Our method is especially useful to study highly polarized cells, such as hippocampal neurons. Scraping and pelleting of such cells to prepare them for cryosectioning destroys contacts between the cell body and neurites and hampers the study of axons and dendrites over longer distances. Another disadvantage of scraping is that neurites are difficult to collect from the dish because they are tightly attached. For cultured primary neurons, an additional complication of the standard method is that the number of cells is usually low, whereas in the conventional procedure considerable amounts of cells are needed to obtain a visible pellet that is manageable for further processing. These disadvantages are overcome with the flat-embedding technique. We developed our method with the principal goal of studying vesicular traffic through axons and dendrites. Because of the highly complex structure of neurons, it is essential for such studies to combine molecular topology with morphological information. On the other hand, molecular characterization of compartments may shed light on their identity and help to define subdomains of the neuron, as shown in Fig 8. Many EM studies in the neurobiology field are performed on Epon or Lowicryl sections, combined with the HRP-DAB staining method or immunogold labeling (e.g.,
We also propose other applications for the flat-embedding technique. A sideways orientation of the gelatin slab on the specimen holder allows sectioning perpendicular to the plane in which cells grow. Thus, oriented sectioning of cells with apical and basolateral subdomains, such as epithelial cells, can be achieved. Flat-embedding can also be of use for non-polarized cells when cellcell contacts or specialized structural adaptations are studied. In the case of non-differentiated PC12 cells, we observed that the plasma membranes were closely aligned along neighboring cells. Such information is lost using the standard procedure. The flat-embedding method provides a means to select a specific cell for cryosectioning. This can be helpful when a restricted subset of cells in a preparation needs to be studied, e.g., after microinjection. Last but not least, the method opens the valuable possibility of direct CLEM using the sensitive and quantitative immunogold labeling of cryosections as an EM approach. We have illustrated this by giving an example using epifluorescence as the light microscopic approach, but clearly this correlative method can be extended to other light microscopic techniques. For example, when antibodies against GFP or its derivatives are used, the procedure is also applicable to performance of CLEM using living cells expressing GFP-conjugated proteins as starting material, providing the exiting possibility of linking cell dynamics to high-resolution information.
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Acknowledgments |
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Supported by grant 805-26-183 from the Life Sciences division of the Dutch Organization of Scientific Research.
We would like to thank Rene Scriwanek and Marc van Peski for the excellent preparation of electron micrographs and for drawing Fig 1, and Dr G. Posthuma for designing the platinum wire device and assistance with Fig 2. We also thank Magda Deneka and Nicolas Barois for their support with the generation of confocal micrographs and our colleagues in the Department of Cell Biology for their insightful comments.
Received for publication September 7, 2001; accepted February 28, 2002.
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