Summary
We have examined the suitability of a variety of fixation regimes for immunofluorescence localization of tubulin and calmodulin in meristematic plant cells. Acrolein and most fixatives that contain glutaraldehyde (GA), while they have been employed by others in immunoenzyme, immunogold or immunofluorescence studies of plant endosperm, animal or plant tissue culture cells, cause unacceptably high background fluorescence of the dense cytoplasm of root meristem cells. Bifunctional imidoester and carbodiimide reagents do not give satisfactory results, either. Fixatives that have produced good results include paraformaldehyde (PFA) with the addition of picric acid or zinc salts or at high pH, a combination of PFA/GA/picric acid, and prefixation in PFA plus a monofunctional imidoester followed by PFA/GA. All of these cross-link the cytoplasm well enough so that cells can withstand isolation procedures, preserve antigenicity, allow antibody penetration and provide good contrast between specific and background fluorescence. The last two fixatives are of particular interest because of the potential they offer for immunoelectron microscopy of densely cytoplasmic, walled cells from tissues.
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References
Aplin JD, Hughes RC (1981) Protein-derivatised glass coverslips for the study of cell-to-substratum adhesion. Anal Biochem 113: 144–148
Bajer AS, Cypher C, Mole-Bajer J, Howard HM (1982) Taxolinduced anaphase reversal: Evidence that elongating microtubules can exert a pushing force in living cells. Proc Natl Acad Sci USA 79: 6569–6573
Berod A, Hartman BK, Pujol JF (1981) Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29: 844–850
Brandtzaeg P (1982) Tissue preparation methods for immunocytochemistry. In:Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol 1. Academic Press, London
Bullock GR (1984) The current status of fixation for electron microscopy: a review. J Microscopy 133: 1–15
Dell'Orto P, Viale G, Columbi R, Braidotti P, Coggi G (1982) Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes. Effect of different fixatives and of proteolytic digestion. J Histochem Cytochem 30: 630–636
De Mey J, Lambert AM, Bajer AS, Moeremans M, De Brabander M (1982) Visualization of microtubules in interphase and mitotic plant cells ofHaemanthus endosperm with the immunogold staining method. Proc Natl Acad Sci USA 79: 1898–1902
—,Moeremans M, Geuens G, Nuydens R, De Brabander M (1981) High resolution light and electron microscopic localization of tubulin with the IGS (immunogold staining) method. Cell Biol Int Rep 5: 889–900
— — — —,Van Belle H, De Brabander M (1981) Immunocytochemical evidence for the association of calmodulin with microtubules of the mitotic apparatus. In:De Brabander M, De Mey J (eds) Microtubules and microtubule inhibitors. Elsevier/North-Holland Biomedical Press, Amsterdam, pp 227–241
Eckert BS, Synder JA (1978) Combined immunofluorescence and high-voltage electron microscopy of cultured mammalian cells, using an antibody that binds to glutaraldehyde-treated tubulin. Proc Natl Acad Sci 75: 334–338
Eldred WD, Zucker C, Karten HJ, Yazulla S (1983) Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry. J Histochem Cytochem 31: 285–292
Galella G, Smith DB (1982) The cross-linking of tubulin with imidoesters. Can J Biochem 60: 71–80
Geiger B, Dutton AH, Tokuyasu KT, Singer SJ (1981) Immunoelectron microscope studies of membrane-microfilament interactions: distributions of α-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol 91: 614–628
Giloh H, Sedat JW (1982) Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates byn-propyl gallate. Science 217: 1252–1255
Grzanna R (1982) Light microscopic immunocytochemistry with fixed, unembedded tissues. In:Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol 1. Academic Press, London, pp 183–204
Hardham AR (1985) Studies on the cell surface of zoospores and cysts of the fungusPhytophthora cinnamomi: the influence of fixation on patterns of lectin binding. J Histochem Cytochem 33: 110–118
Hassell J, Hand AR (1974) Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde. J Histochem Cytochem 22: 223–239
Kendall PA, Polak JM, Pearse AGE (1971) Carbodiimide fixation for immunohistochemistry: observations on the fixation of polypeptide hormones. Experientia 27: 1104–1106
King JC, Lechan RM, Kugel G, Anthony ELP (1983) Acrolein: a fixative for immunocytochemical localization of peptides in the central nervous system. J Histochem Cytochem 31: 62–68
Kondo H (1984) Polyethylene glycol (PEG) embedding and subsequent de-embedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy. J Electron Microsc Tech 1: 227–242
McLean IW, Nakane PK (1974) Periodate-lysineparaformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 22: 1077–1083
Mugnaini E, Dahl A-L (1983) Zinc-aldehyde fixation for lightmicroscopic immunocytochemistry of nervous tissues. J Histochem Cytochem 31: 1435–1438
Osborn M, Weber K (1982) Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. In:Wilson L (ed) Methods in cell biology, vol 24. The cytoskeleton, part A. Academic Press, New York, pp 97–132
Pearse AGE (1972) Histochemistry, theoretical and applied, 3rd edn, vol 2, chap 27, Biogenic amines. Churchill Livingstone, London
— (1980) Histochemistry, theoretical and applied, 4th edn, vol 1, Preparative and optical technology. Churchill Livingstone, London
—,Polak JM (1975) Bifunctional reagents as vapour- and liquidphase fixatives for immunohistochemistry. Histochem J 7: 179–186
Polak JM, Kendall PA, Heath CM, Pearse AGE (1972) Carbodiimide fixation for electron microscopy and immunoelectron cytochemistry. Experientia 28: 368–370
Rieder CL, Bowser SS (1985) Correlative immunofluorescence and electron microscopy on the same section of Epon-embedded material. J Histochem Cytochem 33: 165–171
Schmit A-C, Vantard M, De Mey J, Lambert A-M (1983) Asterlike microtubule centers establish spindle polarity during interphase-mitosis transition in higher plant cells. Plant Cell Rep 2: 285–288
Simmonds DH, Seagull RW, Setterfield G (1985) Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells. J Histochem Cytochem 33: 345–352
Simmonds D, Setterfield G, Brown DL (1983) Organization of microtubules in dividing and elongating cells ofVicia hajastana Grossh. in suspension culture. Eur J Cell Biol 32: 59–66
Smith PF, Keefer DA (1982) Acrolein/glutaraldehyde as a fixative for combined light and electron microscopic immunocytochemical detection of pituitary hormones in immersion-fixed tissue. J Histochem Cytochem 30: 1307–1310
Sofroniew MV, Schrell U (1982) Long-term storage and regular repeated use of diluted antisera in glass staining jars for increased sensitivity, reproducibility, and convenience of single- and two-color light microscopic immunocytochemistry. J Histochem Cytochem 30: 504–511
Somogyi P, Takagi H (1982) A note on the use of picric acidparaformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7: 1779–1783
Stefanini M, De Martino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature (London) 216: 173–174
Takamiya H, Batsford SR, Tokunaga J, Vogt A (1979) Immunohistological staining of antigens on semithin sections of specimens embedded in plastic (GMA-Quetol 523). J Immunol Method 30: 277–288
Tiwari SC, Wick SM, Williamson RE, Gunning, BES (1984) Cytoskeleton and the integration of cellular function in cells of higher plants. J Cell Biol 99: 63s-69s
Tokuyasu KT, Singer SJ (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71: 894–906
Van Lammeren AAM, Keijzer CJ, Willemse MTM, Kieft H (1985) Structure and function of the microtubular cytoskeleton during pollen development inGasteria verrucosa (Mill). H. Duval. Planta 165: 1–11
Vantard M, Lambert A-M, De Mey J, Picquot P, Van Eldik, LJ (1985) Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus. J Cell Biol 101: 488–499
Weber K, Rathke PC, Osborn M (1978) Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy. Proc Natl Acad Sci USA 75: 1820–1824
Webster RE, Henderson D, Osborn M, Weber K (1978) Threedimensional electron microscopical visualization of the cytoskeleton of animal cells: immunoferritin identification of actin- and tubulin-containing structures. Proc Natl Acad Sci USA 75: 5511–5515
Wick SM, Duniec J, 1983: Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Preprophase band development and concomitant appearance of nuclear envelopeassociated tubulin. J Cell Biol 97: 235–243
—,Muto S, Duniec J (1985) Double immunofluorescence labeling of calmodulin and tubulin in dividing plant cells. Protoplasma 126: 198–206
—,Seagull RW, Osborn M, Weber K, Gunning BES (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J Cell Biol 89: 685–690
Willingham MC (1983) An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: the GBS (glutaraldehyde-borohydridesaponin) procedure. J Histochem Cytochem 31: 791–798
—,Wehland J, Klee CB, Richert ND, Rutherford AV, Pastan IH (1983) Ultrastructural immunocytochemical localization of calmodulin in cultured cells. J Histochem Cytochem 31: 445–461
—,Yamada SS (1979) Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells. J Histochem Cytochem 27: 947–960
— —,Pastan I (1980) Ultrastructural localization of tubulin in cultured fibroblasts. J Histochem Cytochem 28: 453–461
Wolosewick J, De Mey J (1982) Localization of tubulin and actin in polyethylene glycol embedded rat seminiferous epithelium. Biol Cell 44: 85–88
— —,Meininger V (1983) Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining. Biol Cell 49: 219–226
Yamamoto N (1984) Immunoelectron microscopic localization of calmodulin with protein A-gold technique. Acta Histochem Cytochem 17: 287–291
— (1985) Immunoelectron microscopic localization of calmodulin in guinea pig testis and spermatozoa. Acta Histochem Cytochem 18: 199–211
—,Yasuda K (1977) Use of a water soluble carbodiimide as a fixing reagent. Acta Histochem Cytochem 10: 14–37
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Wick, S.M., Duniec, J. Effects of various fixatives on the reactivity of plant cell tubulin and calmodulin in immunofluorescence microscopy. Protoplasma 133, 1–18 (1986). https://doi.org/10.1007/BF01293182
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DOI: https://doi.org/10.1007/BF01293182