Synopsis
The methods of fixation and preparation of lymphoid tissues for the immuno-enzyme technique are reviewed. For this technique an enzyme is used first as an antigen and then as a marker to demonstrate its specific antibody. A variety of commonly employed fixatives satisfactorily conserve tissues for the light microscopic detection of antibody but, for electron microscopy, glutaraldehyde or formaldehyde or both are the fixatives of choice. The main technical problem for electron microscopy is to reduce the size of the tissue fragments sufficiently so that the enzymes and their substrates permeate through the fixed tissues. The merits and short-comings of the different preparative techniques are examined and it is shown that the most reproducible results are obtained with 40 μm frozen sections. Some of the problems of non-specific staining arising from fixation procedures, as well as endogenous enzyme activity, are discussed. The evidence for and against antibody inactivation by fixation and enzyme inactivation by interaction with its specific antibody is reviewed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Avrameas, S. (1970). Immuno-enzyme techniques: enzymes as markers for the localisation of antigens and antibodies.Int. Rev. Cytol. 27, 349–85.
Avrameas, S. &Leduc, E. H. (1970). Detection of simultaneous, antibody synthesis in plasma cells and specialised lymphocytes in rabbit lymph nodes.J. exp. Med. 131, 1137–68.
Avrameas, S. &Lespinats, G. (1967). Detection d'anticorps dans des cellules immunocompétentes d'animaux immunisés avec des enzymes.Compt. Rend. Acad. Sci. (Paris) 265, 302–4.
Avrameas, S., Taudou, B. &Ternynck, T. (1971). Specificity of antibodies synthesised by immunocytes as detected by immunoenzyme techniques.Int. Arch. Allergy Appl. Immunol. 40, 161–70.
Avrameas, S. &Ternynck, T. (1969). The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbants.Immunochemistry 6, 53–66.
Graham, R. C., Jr. &Karnovsky, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney; ultrastructural cytochemistry by a new technique.J. Histochem. Cytochem. 14, 291–302.
Hay, J. B., Murphy, M. J., Morris, B. &Bessis, M. C. (1972). Quantitative studies on the proliferation and differentiation of antibody-forming cells in lymph.Am. J. Path. 66, 1–24.
Hess, M. W., Sordat, B., Stoner, R. D. &Cottier, H. (1971). Quantitative titration of antihorseradish peroxidase antibody in mouse serum.Immunochemistry 8, 509–15.
Hugon, J. &Borgers, M. (1966). A direct lead method for the electron microscopic visualisation of alkaline phosphatase activity.J. Histochem. Cytochem. 14, 429–31.
Kraehenbuhl, J. P. &Campiche, M. A. (1969). Early stages of intestinal absorption of specific antibodies in the newborn. An ultrastructural, cytochemical, and immunological study in the pig, rat and rabbit.J. Cell Biol. 42, 345–65.
Kraehenbuhl, J. P., De Grandi, P. B &Campiche, M. A. (1971). Ultrastructural localisation of intracellular antigen using enzyme-labelled antibody fragments.J. Cell Biol. 50, 432–45.
Kuhlmann, W. D. &Avrameas, S. (1971). Glucose oxidase as an antigen marker for light and electron microscopic studies.J. Histochem. Cytochem. 19, 361–8.
Kuhlmann, W. D. &Miller, H. R. P. (1971). A comparative study of the techniques for ultrastructural localisation of anti-enzyme antibodies.J. ultrastruct. Res. 35, 370–85.
Leduc, E. H., Avrameas, S. &Bouteille, M. (1968). Ultrastructural localisation of antibody in differentiating plasma cells.J. exp. Med. 127, 109–18.
Leduc, E. H., Scott, G. B. &Avrameas, S. (1969). Ultrastructural localisation of intracellular immune globulins in plasma cells and lymphoblasts by enzyme-labelled antibodies.J. Histochem. Cytochem. 17, 211–24.
Miller, H. R. P. (1970). A combined light and electron microscopic technique for the localisation of antibody producing cells in the stimulated lymph node. 7th Int. Congr. Electron Microscopy, Vol. I, 537–8.
Miller, H. R. P. &Avrameas, S. (1971). Association between macrophages and specific antibody producing cells.Nature (New Biol.) 229, 184–5.
Miller, H. R. P., Avrameas, S. & Ternynck, T. (1972). Differences between antibody-forming cells responding to primary and to secondary stimulation with horseradish peroxidase.Immunology, ms. submitted for publication.
Scott, G. B. &Avrameas, S. (1968). Intracellular antibody formation demonstrated on ultrathin frozen sections with alkaline phosphatase used as an antigen and as a marker.Electron Microsc. Proc. 4th Eur. Reg. Conf. 2, 201–2.
Scott, G. B., Avrameas, S. &Bernhard, W. (1968). Etude au microscope électronique de la formation d'anticorps à l'aide de phosphatase alcaline utilisée comme antigène.Compt. Rend. Acad. Sci. (Paris) 266, 746–8.
Sordat, B., Sordat, M., Hess, M. W., Stoner, R. D. &Cottier, H. (1970). Specific antibody within germinal centre cells of mice after primary immunisation with horseradish peroxidase: a light and electron microscopic study.J. exp. Med. 131, 77–91.
Straus, W. (1968). Cytochemical detection of antibody to horseradish peroxidase in spleen and lymph nodes.J. Histochem. Cytochem. 16, 237–48.
Straus, W. (1970a). Localisation of antibody to horseradish peroxidase in popliteal lymph nodes of rabbits during the primary and early secondary responses.J. Histochem. Cytochem. 18, 120–30.
Straus, W. (1970b). Localisation of the antigen in popliteal lymph nodes of rabbits during the formation of antibodies to horseradish peroxidase.J. Histochem. Cytochem. 18, 131–42.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Miller, H.R.P. Fixation and tissue preservation for antibody studies. Histochem J 4, 305–320 (1972). https://doi.org/10.1007/BF01005006
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01005006