RAPID COMMUNICATION |
Correspondence to: David H. Hall, Center for C. elegans Anatomy, Dept. of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: hall@aecom.yu.edu
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Summary |
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Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures.
(J Histochem Cytochem 49:949956, 2001)
Key Words: immuno-EM, GFP, microwave fixation, C. elegans, yolk
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Introduction |
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THE NEMATODE Caenorhabditis elegans is a well-known model organism for studies in cell biology and molecular biology (
We have adapted the microwave fixation method (
Yolk and yolk receptor antibodies have been previously employed using standard immunohistochemistry methods to demonstrate the transfer of yolk between the intestine and oocyte of C. elegans (
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Materials and Methods |
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Animals and Antibodies
Wild-type nematodes were cultured according to standard methods (
Microwave Fixation
Animals were rinsed in M9 buffer and placed into a pre-chilled glass well slide. M9 was replaced by a fixative solution consisting of glutaraldehyde and/or paraformaldehyde (Table 1) (Tousimis Research; Rockville, MD) in 0.12 M sucrose, 0.05 mM MgCl2, 0.1 M HEPES, pH 7.2. The specimens were then positioned at a "hotspot" within a Pelco Microwave Oven model 3450 (Ted Pella; Redding, CA) and irradiated at 50% power for 2 min. During this time, the glass well slide was kept chilled by a slurry of ice slush (150 g) and an additional water load of 400 ml was kept within the chamber. The sample volume was approximately 1 ml. Measurements showed that the sample was typically heated from 4C to 10C during this 2-min period. With microwave power off, the sample was allowed to cool for 2 min, during which time the ice slush was replenished. The sample was then irradiated for another 2 min, allowed to chill for 2 min, and irradiated a third time for 2 min. Therefore, total fixation time was only 10 min, with power on for 6 min total. Other microwave regimens were compared (e.g., 4 min on, 2 min off, 4 min on), but longer regimens caused excessive sample heating. After 10 min in fixative, samples were washed several times in 0.12 M sucrose in 0.1 M HEPES, pH 7.2, buffer, and embedded in agarose.
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Embedding in Agarose and LR Gold
Clusters of fixed animals were grouped together in 2.5% SeaPlaque agarose (Sigma; St Louis, MO) near its gelling temperature (30C) and allowed to chill and set (usually overnight) at 4C. Small agarose (2 mm3) blocks were then cut out to carry individual clusters through dehydration and embedding. Specimens were dehydrated through alcohols and into resin according to the schedule in Table 2. After embedment in LR Gold (Polyscience; Warrington, PA) plus 0.5% benzoin methyl ether accelerator (BME; Polyscience), samples were transferred to gelatin capsules in fresh resin, placed in a Pelco cryobox at approximately -20C, and exposed to UV radiation overnight. Most samples hardened within 15 hr; if necessary, the samples were sometimes exposed to an additional 1224 hr of UV to complete curing.
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Immunogold Labeling on Nickel Mesh Grids
Thin sections (60 nm) of LR Gold-embedded worms were collected on formvar-coated nickel mesh grids (Athene thin bar, 200 mesh; Ted Pella). If necessary, nearby sample sections were prescreened by EM to choose regions of special interest for immunostaining. Mesh grids containing sections were washed six times in 0.01 M glycine in 0.1 M phosphate buffer (PB), pH 7.4, at room temperature (RT), blocked with 0.5% gelatin, 0.01 M glycine, 0.5% nonfat dry milk, 1% normal goat serum in 0.1 M PB, pH 7.4, at 37C for 15 min, and washed once in warmed 0.01 M glycine in PB. Each grid was then inverted onto a small droplet (1030 µl) of dilute primary antibody on Parafilm inside a humidified chamber. Antibody was diluted in 0.5% gelatin in PB and spun in a microfuge before use. After exposure to primary antibody for 1 hr at RT, grids were washed six times in PB at RT (floating on 1 ml of buffer while slowly agitating on a shaker table). Grids were inverted on small droplets of secondary gold-linked antibody (Amersham Auro-Probe; 1:30 dilution in 0.5% gelatin in PB). After exposure to secondary antibody for 1 hr at RT, grids were washed six times in PB, fixed in 2.5% glutaraldehyde in PB for 5 min, washed twice in dH2O, and stained in 2% uranyl acetate, pH 5.1, before examination by EM. Controls included (a) no primary antibody, (b) preimmune serum, if available, (c) positive control, i.e., another primary antibody, or (d) negative control, i.e., anti-GFP antibodies on wild-type sections.
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Results and Discussion |
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Localization of GFP
For postembedding immuno-EM of VIT::GFP transgenic animals, several of the commercially available anti-GFP antibodies work very well, with generally low background (Table 1). In the case of VIT::GFP, some anti-GFP antibodies (Clontech and RDI rabbit polyclonal) remain active on tissues fixed under harsh fixation conditions (2.5% glutaraldehyde) (Table 1), thus allowing good preservation of ultrastructural details (Fig 1A). Similar fixation protocols should be satisfactory for any protein labeled with GFP, except in a few difficult tissues. We have begun to utilize these anti-GFP antibodies to follow the transport of secreted proteins from tissue to tissue (
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When labeled by the postembedding technique, the YP170::GFP protein co-localizes in the same dark-staining organelles as native yolk protein in the intestine (Fig 1A and Fig 1B). Gold label is visible in the pseudocoelom in discrete particles (not membrane-bound) (result not shown for anti-GFP; Fig 2 for anti-YP170) and is taken up into oocytes in small membrane-bound granules (Fig 1C and Fig 2) (cf.
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RME-2 Receptor Localization
Another example of preserved antigenicity with microwave fixation is demonstrated by the localization of a new component of C. elegans yolk metabolism, the RME-2 receptor (
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Microwave Fixation Improves Antigen Preservation
Although tissue preservation after microwave fixation is not as good as after immersion fixation, antigenicity is generally excellent, perhaps because of the very short fixation time employed. At all steps we have endeavored to keep the sample temperature low to reduce damage to the epitope. The most obvious benefit of the microwave technique is that many animals in a sample are treated simultaneously and rapidly, whereas an immersion fixation involves the separate cutting by hand of each animal to provide access through the cuticle. The latter procedure is slow, tedious, and results in very irregular results because some animals are cut open shortly after going into fixation, some are cut open at long times after immersion, and some animals fail to be cut open at all. Because it is desirable to produce pellets containing hundreds of animals for the postembedding procedures, it becomes impossible to produce uniform treatment for all specimens by immersion and hand cutting.
The improvement in infiltration/embedment is most obvious in the preservation of embryos, which are impossible to cut open by hand. Their eggshell provides a second barrier to infiltration of fixative, solvent, or resin. In immersion fixations, the embryos typically produce crushed profiles or holes in the resin block because they do not infiltrate at all. The microwave fixation protocol allows fixatives and subsequent solutions to pass both the mother's cuticle and the embryonic eggshell so that those individual embryos are substantially better preserved (Fig 4B). With this technique, we have successfully labeled cell junctions in early and late embryos (unpublished data).
Our current microwave protocol is still not ideal. Preservation is only marginal for most tissues and is often inadequate for smaller cells and more delicate structures, including neurons and cell membranes. The nematode cuticle still is a barrier to the passage of fixatives and embedments that the microwave treatment alleviates but does not correct entirely. However, the resolution is good enough to recognize most tissues and organelles. Eventually, we expect that fast-frozen tissue may prove to be more suitable for postembedding immunocytochemistry to capture fragile membranes and processes (
Protocols must still be tailored to suit the antigen and tissue(s) of interest. A central issue for immuno-EM is to preserve the epitope of interest while minimizing the loss of ultrastructural details. Every fixation procedure is a compromise and one must determine the best method for each tissue and antigen. Fixative choice is an important part of the equation. Glutaraldehyde, for example, reacts rapidly and irreversibly with proteins, making it the best fixative for ultrastructural preservation. However, for the same reason, its use often results in loss of antigenicity. Formaldehyde, a milder fixative that penetrates tissues more rapidly than glutaraldehyde, is gentler to the epitopes but does not provide very good preservation of ultrastructural details. A combination of formaldehyde and low concentrations of glutaraldehyde is often a good compromise. Individual epitopes are still likely to be affected differently by microwave fixation, dehydration, and embedding. Therefore, one needs to test alternative fixatives and treatments for each new antibody under study, as before. Similarly, some tissues may require different treatment regimens, or different osmolarities, to maximize their preservation. In our experience, it is generally helpful to conduct test fixations on at least three different fixative/buffer combinations when a new antibody for postembedding protocol is explored (
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Acknowledgments |
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Supported by grant RR12596 (NCRR) to the Center For C. elegans Anatomy (to DHH) and by March of Dimes grant FY99-583 (BG and DH).
We are grateful to Tylon Stephney (printing) and to Yongjing Li and Judith Kimble for excellent discussions about fixation protocols.
Received for publication April 5, 2001; accepted May 9, 2001.
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