Summary
Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at ≥0° C, such as “progressive lowering of temperature” (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.
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Abbreviations
- AB:
-
antibodies
- AED:
-
aminoethyldextran
- Au10 :
-
colloidal gold (10 nm diameter)
- BSA:
-
bovine serum albumin
- EtOH:
-
ethanol
- FA:
-
formaldehyde
- GA:
-
glutaraldehyde
- LN2 :
-
liquid nitrogen
- MeOH:
-
methanol
- pA:
-
protein A
- Pipes:
-
piperazine-N,N′-bis(2-ethanesulphonic acid)
- PLT:
-
progressive lowering of temperature
- PVP:
-
polyvinylpyrrolidone
- UA:
-
uranylacetate
References
Adoutte A (1988) Exocytosis: biogenesis, transport and secretion of trichocysts. In: Görtz HD (ed) Paramecium. Springer, Berlin, Heidelberg New York, pp 325–363
Adoutte A, Garreau de Loubresse N, Beisson J (1984) Proteolytic cleavage and maturation of the crystalline secretion products of Paramecium. J Mol Biol 180:1065–1081
Armbruster BL, Garavito RM, Kellenberger E (1983) Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique. J Histochem Cytochem 31:1380–1384
Bendayan M, Roth J, Perrelet A, Orci L (1980) Quantitative immunocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell. J Histochem Cytochem 28:149–160
Boonstra J, Van Maurik P, Verkleij AJ (1987) Immunogold labelling of cryosections and cryofractures. In: Steinbrecht RA, Zierold K (eds) Cryotechniques in biological electron microscopy. Springer, Berlin Heidelberg New York, pp 216–230
Carlemalm E, Villiger W (1989) Low temperature embedding. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol. 4. Academic Press, London, pp 29–45
Carlemalm E, Gravito RM, Villiger W (1982) Resin development for electron microscopy and an analysis for embedding at low temperature. J Microsc 126:123–143
Carlemalm E, Villiger W, Hobot JA, Acetarin JD, Kellenberger E (1985) Low temperature embedding with Lowicryl resins: two new formulations and some applications. J Microsc 140:55–63
Erickson PA, Anderson DH, Fischer SK (1987) Use of uranyl acetate en bloc to improve tissue preservation and labeling for post-embedding immunoelectron microscopy. J Electron Microsc Techn 5:303–314
Geuze JJ, Slot JW, Tokuyasu KT (1979) Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex. J Cell Biol 82:697–707
Glas-Albrecht R, Plattner H (1990) High yield isolation procedure for intact secretory organelles (trichocysts) from different Paramecium tetraurelia strains. Eur J Cell Biol 53:164–172
Glas-Albrecht R, Németh A, Plattner H (1990) Secretory proteins and glycoproteins from Paramecium cells. Eur J Protistol 26:149–159
Griffiths G, McDowall A, Back R, Dubochet J (1984) On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res 89:65–78
Humbel B, Müller M (1985) Freeze substitution and low temperature embedding, In: Müller M, Becker RP, Boyde A, Wolosewick JJ (eds) The science of biological specimen preparation. Scanning electron microscopy Inc, AMF O'Hare, Chicago, pp 175–183
Humbel BM, Schwarz H (1989) Freeze-substitution for immunocytochemistry. In: Verkleij AJ, Leunissen JLM (eds) Immunogold labeling in cell biology. CRC Press, Boca Raton, Fla., pp 115–134
Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A
Kellenberger E (1987) The response of biological macromolecules and supramolecular structures to the physics of specimen cryopreparation. In: Steinbrecht RA, Zierold K (eds) Cryotechniques in biological electron microscopy. Springer, Berlin Heidelberg New York, pp 35–63
Kellenberger E, Dürrenberger M, Villiger W, Carlemalm E, Wurtz M (1987) The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J Histochem Cytochem 35:959–969
Knoll G, Oebel G, Plattner H (1982) A simple sandwich-cryogenjet procedure with high cooling rates for cryofixation of biological materials in the native state. Protoplasma 111:161–176
Knoll G, Burger KNJ, Bron R, Van Meer G, Verkleij AJ (1988) Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections. J Cell Biol 107:2511–2521
Knoll G, Braun C, Plattner H (1991) Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure and calcium requirements revealed after rapid mixing and rapid freezing of intact cells. J Cell Biol 113:1295–1304
Luft JH (1973) Embedding media — old and new. In: Koehler JK (ed) Advanced techniques in biological electron microscopy. Springer, Berlin Heidelberg New York, pp 1–34
Monaghan P, Robertson D (1990) Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry. J Microsc 158:355–363
Müller M, Marti T, Kriz S (1980) Improved structural preservation by freeze-substitution. In: Brederoo P, De Priester W (eds) Proc 7th Eur Congr Electron Microscopy. Seventh Eur Congr Electron Microsc Foundation, Leiden, pp 720–721
Murata F, Suzuki S, Tsuyama S, Suganuma T, Imada M, Furihata C (1985) Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach. Histochem J 17:967–980
Peterson JB, Heuser JE, Nelson DL (1987) Dissociation and reassociation of trichocyst proteins: biochemical and ultrastructural studies. J Cell Sci 87:3–25
Plattner H, Bachmann L (1982) Cryofixation: a tool in biological ultrastructural research. Int Rev Cytol 79:237–304
Plattner H, Schmitt-Fumian WW, Bachmann L (1973) Cryofixation of single cells by spray-freezing. In: Benedetti EL, Favard P (eds) Freeze-etching. Techniques and applications. Soc Franç Microsc Electronique, Paris, pp 81–100
Plattner H, Stürzl R, Matt H (1985) Synchronous exocytosis in Paramecium cells. IV. Polyamino-compounds as potent trigger agents for repeatable trigger-redocking cycles. Eur J Cell Biol 36:32–37
Posthuma C, Slot JW, Geuze HJ (1987) Usefulness of the immunogold technique in quantitation of a soluble protein in ultrathin sections. J Histochem Cytochem 35:405–410
Pscheid P, Schudt C, Plattner H (1981) Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments. J Microsc 121:149–167
Rauh J, Nelson DL (1981) Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia. J Cell Biol 91:860–865
Roth J (1986) Post-embedding cytochemistry with gold-labelled reagents: a review. J Microsc 143:125–137
Roth J, Taatjes DJ, Warhol MJ (1989) Prevention of non-specific interactions of gold-labeled reagents on tissue sections. Histochemistry 92:47–56
Sitte H, Edelmann L, Neumann K (1987) Cryofixation without pretreatment at ambient pressure. In: Steinbrecht RA, Zierold K (eds) Cryotechniques in biological electron microscopy. Springer, Berlin Heidelberg New York, pp 87–113
Steers E, Beisson J, Marchesi VT (1969) A structural protein extracted from the trichocyst of Paramecium aurelia. Exp Cell Res 57:392–396
Steinbrecht RA, Müller M (1987) Freeze-substitution and freeze-drying. In: Steinbrecht RA, Zierold K (eds) Cryotechniques in biological electron microscopy. Springer, Berlin Heidelberg New York, pp 149–172
Stierhof YD, Schwarz H, Frank H (1986) Transverse sectioning of plastic-embedded immunolabeled cryosections: morphology and permeability to protein A-colloidal gold complexes. J Ultrastruct Mol Struct Res 97:187–196
Stierhof YD, Schwarz H, Dürrenberger M, Villiger W, Kellen-berger E (1991) Yield of immunolabel compared to resin sections and thawed cryosections. In: Hayat MA (ed) Colloidal gold: principles, methods and applications, vol 3. Academic Press, New York, pp 87–115
Stirling JW (1990) Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques. J Histochem Cytochem 38:145–157
Tindall SH, Devito LD, Nelson DL (1989) Biochemical characterization of the proteins of Paramecium secretory granules. J Cell Sci 92:441–447
Tokuyasu KT (1978) A study of positive staining of ultrathin frozen sections. J Ultrastruct Res 63:287–307
Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381–403
Usuda M, Ma H, Hanai T, Yokota S, Hashimoto T, Nagata T (1990) Immuno-electron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes. J Histochem Cytochem 38:617–623
Van Ewijk W, Van Soest PL, Verkerk A, Jongkind JF (1984) Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry. Histochem J 16:179–193
Verkleij AJ, Leunissen JLM eds (1989) Immuno-gold labeling in cell biology. CRC Press, Boca Raton, Fla.
Zasadzinski JAN (1988) A new heat transfer model to predict cooling rates for rapid freezing fixation. J Microsc 150:137–149
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Bittermann, A.G., Knoll, G., Németh, A. et al. Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Histochemistry 97, 421–429 (1992). https://doi.org/10.1007/BF00270389
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DOI: https://doi.org/10.1007/BF00270389