RAPID COMMUNICATION |
Preservation of Immunoreactivity and Fine Structure of Adult C. elegans Tissues Using High-pressure Freezing
Biologie Cellulaire de la Synapse, École Normale Supérieure, Paris, France (PR,RMW,AT,J-LB); and Department of Biology, University of Utah, Salt Lake City, Utah (EMJ)
Correspondence to: Jean-Louis Bessereau, Biologie Cellulaire de la Synapse, École Normale Supérieure, 46 rue d'Ulm, Paris 75005 France. E-mail: jlbesse{at}wotan.ens.fr
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Summary |
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Key Words: C. elegans immunoelectron microscopy high-pressure freezing immunogold
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Introduction |
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For classical electron microscopy studies in C. elegans, such as those conducted to describe its anatomy (Epstein et al. 1974; Waterston et al. 1974
; Ward et al. 1975
; Albertson and Thomson 1976
; White et al. 1976
,1986
), best preservation and embedding is obtained from animals that have been cut to allow chemical fixatives and embedding solutions access to inner tissues. In combination with strong fixative reagents, such as osmium tetroxide, excellent preservation for ultrastructural studies is achieved; however, immunoreactivity is lost. If weaker fixative reagents are used, such as paraformaldehyde, proteins often remain antigenic (Park et al. 2001
) but structural preservation is compromised. Recent attempts to remedy this paradox in C. elegans immunoelectron microscopy have taken two approaches: increase cuticle permeability or physically immobilize tissues before chemical fixation.
To increase cuticle permeability, Paupard et al. (2001) exposed intact animals to microwaves. The rate of diffusion after exposure to microwaves was sufficient to preserve gross cell structure and protein immunoreactivity. However, fine structures, such as those in neurons, were not well preserved (Paupard et al. 2001
).
To physically immobilize tissues before chemical fixation, Kirkham et al. (2003) froze C. elegans embryos under high pressure and then chemically fixed the specimens at low temperatures, a strategy recently used for morphological studies in C. elegans (Dernburg et al. 1998
; WilliamsMasson et al. 1998
; Rappleye et al. 1999
; Howe et al. 2001
; Koppen et al. 2001
; MacQueen et al. 2002
; Rolls et al. 2002
) and for immunoelectron microscopy studies in other organisms (reviewed in McDonald 1999
). Freezing under high pressure is capable of solidifying water rapidly as vitreous ice, an amorphous state of water, which physically preserves tissues. Chemical tissue fixation and embedding can then occur over an extended period of time without deterioration of the tissue ultrastructure. In the embryo, overall cell structure as well as protein immunoreactivity were well preserved after high-pressure freezing. In addition, high-pressure freezing has been used to detect cuticle antigens in dauer arrested larvae (Favre et al. 1995
,1998
). These results suggested that a similar method might be suitable for studies in the adult.
Here we describe a protocol for the preservation of adult tissues in C. elegans for immunoelectron microscopy studies. Specifically, living animals are immobilized by high-pressure freezing, then chemically fixed, dehydrated, and embedded at low temperatures over an extended period of time. With this protocol, cell morphology is well preserved, including fine structures such as microtubules and actinmyosin lattices. In addition, protein immunoreactivity is preserved. We successfully stained ultrathin sections with antibodies directed against components of fibrous organelles, cell adhesion junctions, and synaptic vesicles, and an exogenous GFP fusion protein expressed in neurons. This protocol therefore provides a method for localization of endogenous and exogenous proteins with respect to subcellular compartments in most, if not all, C. elegans tissues.
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Materials and Methods |
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High-pressure Freeze Immobilization
N2 Bristol or EG1399[lin-15(n765)X; oxEx81(Pacr-5::GAP-43-GFP, lin-15(+))] (Knobel et al. 2001) worms were cultured under standard conditions (Brenner 1974
) at 20C and young adult hermaphrodites were immobilized in E. coli by high-pressure freezing. Specifically,
10 animals were placed in a specimen chamber of 100-µm depth (Bal-Tec, Liechtenstein; part number LZ02135VN A and B) filled with E. coli (OP50) and frozen rapidly at -176C under high pressure (P >2100 bar) in a Bal-Tec HPM010 apparatus according to the manufacturer's recommendation. Chambers containing frozen specimens were stored in liquid nitrogen.
Chemical Fixation and Freeze-substitution
Frozen animals were chemically fixed and dehydrated in a Reichart AFS apparatus (Leica; Oberkochen, Germany). Specimen chambers were moved into either anhydrous acetone (Carlo Erba; Rodano, Italy) containing 0.01% osmium tetroxide (Polyscience) or anhydrous methanol (Carlo Erba) containing 4% paraformaldehyde (Riedel-de Haën; Seelze, Germany) and 1.5% uranyl acetate (Sigma; St Louis, MO) at -90C. Once placed in the cold fixative solution, the specimen chamber lids were removed. Samples were incubated for 7 days with one change of solution midway through incubation. Frozen EG1399 animals were incubated with anhydrous methanol containing 1.5% uranyl acetate without paraformaldehyde.
Embedding
Animals fixed in osmium were washed in anhydrous acetone then embedded in Araldite (Ernest F. Fullam; Latham, NY) and those fixed in paraformaldehyde were washed in anhydrous methanol, then embedded in Lowicryl HM20 (Polysciences; Warrington, PA) in the following manner. First, the temperature in the Reichart AFS was progressively raised to -45C in increments of 4C/hr. Specimen chambers were placed in wash solution and the frozen galette of worm and bacteria was removed by either suction from a pipette or scraping with a needle. Galettes were further washed with 10-min incubations until the solution remained clear (at least three times) before infiltration. For embedding in Araldite, the galettes were slowly warmed to 20C by incubation at -20C overnight, -4C for 1 hr, and room temperature (RT) for 1 hr, then placed in a Beem capsule cap and infiltrated with resin by incubation in 50% Araldite in acetone for 3 hr at 20C, 90% Araldite in acetone overnight at 4C, and twice in pure Araldite at 20C before polymerization at 60C over 48 hr. After osmium fixation, animals appear light brown within the resin block. For embedding in Lowicryl, the galettes were kept at -45C in the Reichart AFS while being moved to a Beem capsule cap and placed in a 1:2 solution of Lowicryl to methanol for 12 hr, then in 2:1 Lowicryl to methanol for 12 hr, then pure Lowicryl for 3 days with one change of Lowicryl. Lowicryl was polymerized by UV exposure for 60 hr. After polymerization the temperature of the embedded galette was raised to RT at a rate of 6.5C/hr. After paraformaldehyde fixation animals appear opaque.
Mounting Galettes for Sectioning
Once polymerized, the galettes were examined under a dissecting microscope to identify an appropriate sectioning path, split by cutting with a razor blade to isolate worms to be sectioned, and glued to an Araldite block with Superglue for sectioning. Typically, several worms can be mounted from an individual galette on independent blocks and sectioned in a desired orientation.
Antibodies
The mouse monoclonal antibodies MH27 (Francis and Waterston 1991; an anti-AJM-1 antibody (Koppen et al. 2001
) and anti-UNC-17 (Lickteig et al. 2001
) were diluted 1:200 and 1:10, respectively. Rabbit polyclonal antisera AF2 (an anti-VAB-10A antibody; Bosher et al. 2003
) and Molecular Probes (Eugene, OR) a11122 (an anti-GFP antibody) were diluted 1:2000 and 1:200, respectively.
Immunogold Labeling
Thin (40- or
80-nm) sections of Lowricryl-embedded worms were collected on formvar-coated nickel slot grids (Pelanne Instruments; Toulouse, France). For staining with MH27, AF2 and a11122 antibodies, sections on slot grids were blocked in a 5% BSA Cohn Fraction V (Research Organics; Cleveland, OH) PBST (PBS, pH 7.4, with 0.01% Tween-20; Merck, Whitehouse Station, NJ) solution for 30 min at RT then washed three times in 0.1% BSAPBST for 5 min each. Grids were then incubated in a humidified chamber with dilute primary antibody, described above, in 0.01% CWFS gelatin (Aurion; Wageningen, Netherlands) 0.001% Tween-20PBS for 1 hr at RT, washed six times in 0.1% BSAPBST, incubated in a humidified chamber with goat anti-rabbit or anti-mouse 10- or 15-nm gold-conjugated antibodies (BBInternational; Cardiff, UK) diluted 1:200 in 0.01% gelatin0.001% Tween-20PBS. After exposure to secondary antibody for 30 min, grids were washed six times in 0.1% BSAPBST, three times in PBS, fixed in 2% glutaraldehyde (TAAB; Poole, UK) PBS then washed once in PBS and twice in dH2O.
For staining with the anti-UNC-17 antibody, sections on slot grids were blocked in a 1% goat serum (Aurion) for 30 min at RT, then washed three times in PBS for 5 min each. Grids were then incubated in a humidified chamber with dilute primary antibody in 0.5% CWFS gelatin (Aurion)0.001% Tween-20PBS for 1 hr at RT, washed six times in PBS, incubated in a humidified chamber with goat anti-mouse 10-nm gold conjugated antibodies (BBInternational) diluted 1:50 in 0.5% gelatin0.001% Tween-20PBS. After exposure to secondary antibody for 30 min, grids were washed nine times in PBS, fixed in 2% glutaraldehydePBS, then washed once in PBS and twice in dH2O. Unless otherwise stated, the specificity of staining was judged by the signal-to-noise ratio for each primary antibody, which was calculated by dividing the bead density in specified tissues by the density within neighboring E. coli on the section.
Counterstaining
Before examination, some sections were counterstained by incubating with 2.5% uranyl acetate in 70% methanol for 4 min, followed by washing and incubating with lead citrate (0.15 M lead nitrate, 0.12 M sodium citrate in CO2-free dH2O) for 2 min.
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Results |
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We observed a similar level of preservation throughout the animal after high-pressure freezing (Figure 1) . Specifically, in the seam cells membranous structures are well preserved, such as the mitochondrial and endoplasmic reticulum, along with the neighboring excretory canal (Figure 1A). In the intestine, mitochondria, endoplasmic reticulum, and plasma membranes are well preserved (Figures 1B and 1E). Likewise, muscle and epidermal morphology is well preserved (Figures 1C and 1F). The organization of the contractile apparatus remains intact, including the alignment of the dense bodies, M-line, and fibrous organelles. In addition, fine structure within neurons, such as microtubules and synaptic vesicles, is well preserved (Figures 1D and 1G).
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Discussion |
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Classical Fixation vs High-pressure Freeze Immobilization
Classical ultrastructural analysis of the C. elegans anatomy requires the breaching of the animal's cuticle by cutting to allow access of chemical fixatives and embedding resins into the interior tissues. Because the body cavity of C. elegans is slightly pressurized, breaching of the animal's cuticle before fixation has the potential to introduce structural artifacts, such as shrinkage of the extracellular space. In addition, the solutions typically used for classical fixation are hyperosmotic, which can exacerbate tissue contraction and membrane ruffling. By contrast, high-pressure freezing arrests tissues in a vitrified state. The vitrified state increases the time window for solutions to penetrate the cuticle and preserve interior tissues.
The importance of this point for morphological studies is illustrated by comparing the morphology of the nerve cord preserved via high-pressure freezing to that which was preserved via classical techniques (Figure 2). After classical techniques, neuron processes are irregularly shaped, with very little space between the processes. However, after high-pressure freezing the neuron processes are round, with regularity in shape and space between processes. It is interesting to note that the average size of membrane structures, such as synaptic vesicles and mitochondria, does not differ between fixation protocols. This suggests that the round appearance of neurons is not due to general swelling of tissues but rather to better preservation of cell morphology by fixation after high-pressure freezing. These observations are consistent with the notion that the morphology observed after high-pressure freezing closely represents the true morphology of tissues in the living animal.
However, membranes appear to be better stained with classical fixation compared to our current fixation protocol. This is probably due to slight differences in the chemistry during fixation at -90C or above freezing. We are now testing whether different concentrations of osmium, various blends of methanol and acetone, or other heavy ions may improve the staining of membranes at very low temperatures.
Morphology of Tissues in Araldite vs Lowicryl
The morphological preservation observed after fixation with osmium tetroxide and embedding in Araldite is outstanding, but both osmium tetroxide and Araldite are incompatible with most immunolabeling. In C. elegans, only two cuticle antigens have been labeled thus far using such a protocol (Favre et al. 1995,1998
). We failed to observe any staining of osmium-preserved tissues with antibodies that worked using alternative sample processing protocols (PR and RW; data not shown). To circumvent this problem, we substituted paraformaldehyde for osmium tetroxide and Lowicryl for Araldite. At a gross level, the morphology of preserved tissues is similar between the two protocols. For example, overall cell integrity is maintained in all tissues, and intracellular structures, such as endoplasmic reticulum and mitochondria, are preserved. However, on close inspection differences are observed between the two methods of preservation. Specifically, in the samples processed with paraformaldehyde and Lowicryl, membranes appear slightly fuzzy in all tissues, the organization of the muscle contractile apparatus, particularly the M-line, is not maintained to the same extent, and the continuity of membranous structures is variable compared to the osmium-fixed tissues. These differences are likely due to the fact that Lowicryl HM20 is a soft resin that is difficult to cut and that paraformaldehyde is not as strong a fixative as osmium, especially at these low temperatures. However, at least for the nerve ring, paraformaldehye appears to be dispensable for ultrastructural preservation of high-pressure freeze immobilized tissues. For example, the micrographs shown in Figure 7 are of neuron profiles preserved with methanol fixation alone. Despite the differences between Lowicryl- and Araldite-embedded samples, the level of structural preservation is sufficient for protein localization studies with relation to fine subcellular structures in most tissues, including neurons.
Reproducibility of Morphological Preservation
The usefulness of any technique depends on its reproducibility. We have repeated the paraformaldehyde fixation procedure outlined above a number of times to determine its reproducibility. Thus far, we have observed that the quality of tissue preservation may vary among experiments and even within the same sample. These differences are likely due to small and uncontrolled variability that occurs during the freezing step. Our current solution to this problem involves the freezing and processing of several samples in parallel, followed by the examination of all samples to identify an animal in which the morphology has been well preserved. This animal is then used for subsequent studies. It is interesting to note, however, that every paraformaldehyde-fixed sample tested has retained immunoreactivity, suggesting that whereas morphology may be difficult to preserve, immunoreactivity preservation is robust under these conditions.
Immunolabeling
To date, we have tested and observed immunogold labeling using both monoclonal and polyclonal antibodies generated in mouse and rabbit that recognize antigens present in muscle, epidermis, intestine, and neurons. These successes suggest that this method will be applicable to a number of studies. However, not surprisingly, a few antibodies tested so far (2/7 rabbit antibodies) do not appear to recognize their epitope in sections, which could be due to epitope destruction or masking or due to the staining condition. Therefore, each antibody will need to be tested in an empirical fashion for immunoreactivity on sections. In addition, amplification techniques, such as silver enhancement (Humbel et al. 1995), should be compatible with our fixation/embedding protocol and may aid in the detection of low-abundance proteins. Protein tags, such as GFP, have the potential to alleviate this problem. Once staining conditions are optimized for a particular set of epitope and antibody, these conditions could be used to study the localization of countless tagged proteins.
Application and Potential Uses in Other Systems
The full potential of this techniquecryoimmobilization, weak chemical fixation, and embeddingresides in the fact that proteins are immunolabeled on ultrathin sections after tissue embedding. Therefore, many antibodies and conditions can be tested on sections from the same block of preserved tissue. This eliminates variability in staining due to variation among fixations. In addition, resin-embedded tissues can be maintained over an extended period, making it feasible to accumulate a collection of preserved samples. In the context of a genetic model organism, such as C. elegans, one can imagine a library of mutants in embedding blocks. By staining sections from the library, the effects of disrupting a specific protein or pathway on subcellular domains can be characterized at the level of localization of a large number of proteins. Similar tissue libraries could also be constructed of other model organisms, such as Drosophila, or of vertebrate tissues, thus accelerating the characterization of protein localization relative to well-preserved subcellular structures in these systems as well.
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Acknowledgments |
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We thank Kent McDonald and Claude Anthony for kindly sharing many protocols, Jean-Pierre Lechaire and the EM facility of IFR 83 (Paris) for the use of their Bal-Tec HPF, Warren Davis for the micrograph in Figure 2, Michel Labouesse for the anti-VAB-10A antibody, the NICHD for the MH27 antibody, Jim Rand for the anti-UNC-17 antibody, and Marie Delattre for contributing the micrographs presented in Figure 3.
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Footnotes |
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Received for publication June 30, 2003; accepted August 27, 2003
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