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Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens

Published online by Cambridge University Press:  19 November 2013

Kent L. McDonald
Kent L. McDonald*
Affiliation:
Electron Microscope Laboratory, University of California, Berkeley, CA 94720, USA
*
*Corresponding author. E-mail: [email protected]
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Abstract

A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5–2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

Keywords

electron microscopybiological specimen preparationresin infiltrationresin polymerizationcorrelative light and electron microscopyLR Whitehigh pressure freezingfreeze substitutionon-section immunolabelingrapid embedding methods
Type
Techniques, Software, and Instrumentation Development
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 152 - 163
Copyright
Copyright © Microscopy Society of America 2014 

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References

Bain, J.M. & Gove, D.W. (1971). Rapid preparation of plant tissues for electron microscopy. J Microsc 93, 159–162.CrossRefGoogle Scholar
Boutté, Y., Jonsson, K., McFarlane, H.E., Johnson, E., Gendre, D., Swarup, R., Friml, J., Samuels, L., Robert, S. & Bhalerao, R.P. (2013). ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation. Proc Natl Acad Sci (USA) 110, 16259–16264.Google Scholar
Bozzola, J.J. & Russell, L.D. (1999). Electron Microscopy: Principles and Techniques for Biologists, 2nd ed. Salisbury, MA: Jones and Bartlett.Google Scholar
Coulter, H.D. (1967). Rapid and improved methods for embedding biological tissues in Epon 812 and Araldite 502. J Ultrastruct Res 20, 346–355.Google Scholar
Craig, S., Gilkey, J.C. & Staehelin, L.A. (1987). Improved specimen support cups and auxiliary devices for the Balzers high pressure freezing apparatus. J Microsc 48, 103–106.CrossRefGoogle Scholar
Dubochet, J. (2007). The physics of rapid cooling and its implications for cryoimmobilization of cells. Meth Cell Biol 79, 7–21.Google Scholar
Ellisman, M.H., Deerinck, T.J., Shu, X. & Sosinsky, G.E. (2012). Picking faces out of a crowd: Genetic labels for identification of proteins in correlated light and electron microscopy imaging. Meth Cell Biol 111, 139–155.CrossRefGoogle ScholarPubMed
Falbel, T.G., Koch, L.M., Nadeau, J.A., Segui-Simarro, J.M., Sack, F.D. & Bednarek, S.Y. (2003). SCD1 is required for cell cytokinesis and polarized cell expansion in Arabidopsis thaliana . Development 130, 4011–4024.CrossRefGoogle ScholarPubMed
Giberson, R.T. & Demaree, R.S. Jr. (Eds.) (2001). Microwave Techniques and Protocols. Totowa, NJ: Humana Press.Google Scholar
Giberson, R.T., Smith, R.L. & Demaree, R.S. (1995). Three hour microwave tissue processing for transmission electron microscopy: From unfixed tissues to sections. Scanning 17(Suppl 5), 26–27.Google Scholar
Glauert, A.M. (1975). Fixation, dehydration and embedding of biological specimens. In Practical Methods in Electron Microscopy, vol. 3, Part 1, Glauert, A.M. (Ed.). Amsterdam: North-Holland.Google Scholar
Hawes, P., Netherton, C.L., Mueller, M., Wileman, T. & Monaghan, P. (2007). Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc 226, 182–189.CrossRefGoogle ScholarPubMed
Hayat, M.A. (1986). Glutaraldehyde: Role in electron microscopy. Micron Microsc Acta 17, 115–135.CrossRefGoogle Scholar
Hayat, M.A. (2000). Principles and Techniques of Electron Microscopy, 4th ed. Boca Raton, FL: CRC Press.Google Scholar
Hayat, M.A. & Giaquinta, R. (1970). Rapid fixation and embedding for electron microscopy. Tissue and Cell 2, 191–195.Google Scholar
Hess, M.W. (2007). Cryopreparation methodology for plant cell biology. Meth Cell Biol 79, 57–100.CrossRefGoogle ScholarPubMed
Hurbain, I. & Sachse, M. (2011). The future is cold: Cryo-preparation methods for transmission electron microscopy of cells. Biol Cell 103, 405–420.Google Scholar
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A. (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol 192, 111–119.Google Scholar
Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M. & Briggs, J.A.G. (2012). Precise, correlated fluorescence microscopy and electron microscopy of Lowicryl sections using fluorescent fiducial markers. Meth Cell Biol 111, 235–257.Google Scholar
Leunissen, J.L. & Yi, H. (2009). Self-pressurized rapid freezing (SPRF): A novel cryofixation method for specimen preparation in electron microscopy. J Microsc 235, 25–35.Google Scholar
Marchese, S.P. & Johnson, S.P.S. (1982). A simple method for the progressive infiltration of resin into a dehydrated biological sample. Proc Roy Microsc Soc 17, 311.Google Scholar
McCully, M.E. & Canny, M.J. (1985). The stabilization of labile configurations of plant cytoplasm by freeze-substitution. J Microsc 139, 27–33.Google Scholar
McDonald, K. (2007). Cryopreparation methods for electron microscopy of selected model systems. Meth Cell Biol 79, 23–56.Google Scholar
McDonald, K., Schwarz, H., Mueller-Reichert, T., Webb, R., Buser, C. & Morphew, M. (2010). “Tips and tricks” for high pressure freezing of model systems. Meth Cell Biol 96, 671–694.Google Scholar
McDonald, K.L. (1999). High pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. In Electron Microscopy Methods and Protocols, Hajibagheri, M.A.N. (Ed.), pp. 77–97. Totowa, NJ: Humana Press.CrossRefGoogle Scholar
McDonald, K.L. & Webb, R.I. (2011). Freeze substitution in 3 hours or less. J Microsc 243, 227–233.Google Scholar
Millonig, G. (1976). Laboratory Manual of Biological Electron Microscopy. Vercelli, Italy: Saviolo.Google Scholar
Möbius, W. (2009). Cryopreparation of biological specimens for immunoelectron microscopy. Ann Anat 191, 231–247.Google Scholar
Mueller-Reichert, T. & Verkade, P. (Eds.) (2012). Correlative Light Electron Microscopy. San Diego: Academic Press.Google Scholar
Müller-Reichert, T., O'Toole, E.T., Hohenberg, H. & McDonald, K.L. (2003). Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J Microsc 212, 71–80.CrossRefGoogle ScholarPubMed
Newman, G.R. & Hobot, J.A. (1993). Resin Microscopy and On-Section Immnocytochemistry. Berlin: Springer-Verlag.Google Scholar
Newman, G.R. & Hobot, J.A. (1999). Resins for combined light and electron microscopy: A half century of development. Histochem J 31, 495–505.Google Scholar
Nicolas, M.-T. & Bassot, J.-M. (1993). Freeze substitution after fast-freeze fixation in preparation for immunocytochemistry. Microsc Res Tech 24, 474–487.Google Scholar
Nixon, S.J., Webb, R.I., Floetenmeyer, M., Schieber, N., Lo, H.P. & Parton, R.G. (2009). A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic 10, 131–136.CrossRefGoogle ScholarPubMed
Ondzighi, C.A., Christopher, D.A., Cho, E.J., Chang, S.-C. & Staehelin, L.A. (2008). Arabidopsis protein disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles before programmed cell death of the endothelium in developing seeds. Plant Cell 20, 2205–2220.Google Scholar
Otegui, M.S., Herdera, R., Schulzec, J., Jungc, R. & Staehelin, L.A. (2006). The proteolytic processing of seed storage in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell 18, 2567–2581.CrossRefGoogle ScholarPubMed
Parton, R.G. (1995). Rapid processing of filter-grown cells for Epon embedding. J Histochem Cytochem 43, 731–733.Google Scholar
Porta, D. & Lopez-Iglesias, C. (1998). A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella . Tissue Cell 30, 368–376.Google Scholar
Reymond, O.L. & Pickett-Heaps, J.D. (1983). A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely orientated sectioning of single cells by light microscopy. J Microsc 130, 79–84.Google Scholar
Reynolds, E.S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17, 208–212.Google Scholar
Robbins, E. & Jentzsch, G. (1967). Rapid embedding of cell culture monolayers and suspensions for electron microscopy. J Histochem Cytochem 15, 181–182.Google Scholar
Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H.H., Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O. & Benfey, P. N. (2005). COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation. Plant Cell 17, 1749–1763.Google Scholar
Rowden, G. & Lewis, M.G. (1974). Experience with a three-hour electron microscopy biopsy service. J Cli Pathol 27, 505–510.CrossRefGoogle ScholarPubMed
Seguı-Simarro, J.M., Austin, J.R. II, White, E.A. & Staehelin, L.A. (2004). Electron tomographic analysis of somatic cell plate formation in meristematic cells of arabidopsis preserved by high-pressure freezing. Plant Cell 16, 836–856.Google Scholar
Shimoni, E., Rav-Hon, O., Ohad, I., Brumfeld, V. & Reich, Z. (2005). Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography. Plant Cell 17, 2580–2586.Google Scholar
Skepper, J.N. (2000). Immunocytochemical strategies for electron microscopy: Choice or compromise. J Microsc 199, 1–36.Google Scholar
Spurr, A.R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastr Res 26, 31–43.CrossRefGoogle ScholarPubMed
Webster, P. (2007). Microwave-assisted processing and embedding for transmission electron microscopy. In Methods in Molecular Biology: Electron Microscopy—Methods and Protocols, Kuo, J. (Ed.), pp. 257–289. Totowa, NJ: Humana Press.Google Scholar
Weibull, C., Villiger, W. & Carlemalm, E. (1984). Extraction of lipids during freeze-substitution of Acholeplasma laidlawii-cells for electron microscopy. J Microsc 134, 213–216.Google Scholar