Summary
Pre-embedding immunohistochemistry with subsequent embedding in hydroxypropyl methacrylate enables one to obtain high resolution staining of antigens in 1 μ tissue sections. A routine method using formaldehyde fixation, methanol permeation, and an indirect method with fluoresceinlabeled second antibody is described. This method is compared with other pre-embedding staining procedures. To illustrate the method the mouse small intestine was chosen as a model and stained with antibodies to tubulin, actin, and fibronectin. Some anticipated and some unusual staining patterns were found.
Similar content being viewed by others
References
Bennett HS, Wyrick AD, Lee SW, McNeil JH (1976) Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol 51: 71–97
Bergroth V, Reitamo S, Konttinen YT, Lalla M (1980) Sensitivity and nonspecific staining of various immunoperoxidase techniques. Histochemistry 68:17–22
Burns J (1975) Background staining and sensitivity of the unlabeled antibody-enzyme (PAP) method. Comparison of the peroxidase labelled antibody sandwich method using formalin fixed paraffin embedded material. Histochemistry 43: 291–294
Carsten ME, Mommaerts WFHM (1963) A study of actin by means of starch gel electrophoresis. Biochemistry 2: 28–32
Carnegie JA, McCully ME, Robertson HA (1980) Embedment in glycol methacrylate at low temperature allows immunofluorescent localization of a labile tissue protein. J Histochem Cytochem 28: 308–310
Craig S, Goodschild DJ, Millerd A (1979) Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue. J Histochem Cytochem 27: 1312–1316
Cleveland DW, Hwo S-Y, Kirschner MW (1977) Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J Mol Biol 116: 227–247
Dustin P (1978) Microtubules. Springer, Berlin Heidelberg New York
Engvall E, Ruoslahti E (1977) Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 20: 1–5
Franklin RM (1981) Pre-embedding staining for immunohistochemistry at the light microscopic level. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol I. Academic Press, London
Franklin RM, Martin M-T (1980a) A novel method to improve immunofluorescence in histological specimens. Eur J Cell Biol 21: 134–138
Franklin RM, Martin M-T (1980b) Staining and histochemistry of undecalcified bone embedded in a water-miscible plastic. Stain Technol 55: 313–321
Franklin RM, Martin M-T, Longato R (1981) A simple procedure for extracting methacrylic acid from water-miscible methacrylates. Stain Technol (in press)
Fulcher RG, Holland AA (1971) Fluorescent antibody staining of 1–2 μm sections of hyphae of Ophiobolus graminis Sacc. embedded in glycol methacrylate. Arch Microbiol 75: 281–284
Gill GW, Frost JK, Miller KA (1974) A new formula for a half-oxidized hematoxylin solution that neither overstains nor requires differentiation. Acta Cytol 18: 300–311
Graham RC Jr, Karnovsky MJ (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
Grzanna R, Molliver ME, Coyle JT (1978) Visualization of central noradrenergic neurons in thick sections by the unlabeled antibody method: A transmitter-specific Golgi image. Proc Natl Acad Sci USA 75: 2502–2506
Humanson GL (1979) Animal tissue techniques 4th Edit. WH Freeman and Co, San Francisco
Jockush BM, Kelley KH, Meyer RK, Burger MM (1978) An efficient method to produce specific anti-actin. Histochemistry 55: 177–184
Johnston NW, Bienenstock J (1974) Abolition of non-specific fluorescent staining of eosinophils. J Immunol Methods 4: 189–194
Osborn M, Weber K (1977) The display of microtubules in transformed cells. Cell 12: 561–571
Pearse AGE (1980) Histochemistry, theoretical and and applied, vol 1: Preparative and optical technology 4th edn. Churchill Livingston, Edinburgh London New York
Petrusz P, Ordronneau P, Finley JCW (1980) Criteria of reliability for light microscopic immunocytochemical staining. Histochem J 12: 333–348
Pickel VM, Joh TH, Reis DJ (1975) Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain. Proc Natl Acid Sci USA 72: 659–663
Polak JM, Pearse AGE, Heath CM (1975) Complete identification of endocrine cells in the gastrointestinal tract using semithin-thin sections to identify motilin cells in human and animal intestine. Gut 16: 225–229
Rees MK, Young M (1967) Studies on the isolation and molecular properties of homogeneous globular actin. Evidence for a single polypeptide chain structure. J Biol Chem 242: 4449–4458
Rodning CB, Erlandsen SL, Coulter HD, Wilson ID (1980) Immunohistochemical localization of IgA antigens in sections embedded in epoxy resin. J Histochem Cytochem 28: 199–205
Rohde H, Wick G, Timpl R (1979) Immunochemical characterization of the basement membrane glycoprotein laminin. Eur J Biochem 102: 195–201
Stearns ME, Brown DL (1979) Purification of microtubule-associated protein based on its preferential association with tubulin during microtubule initiation. FEBS Lett 101: 15–20
Sternberger LA (1980) Immunocytochemistry. 2nd Edit. J. Wiley & Sons, New York
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 Methods 30: 277–288
Takamiya H, Batsford S, Vogt A (1980) An approach to postembedding staining of protein (immunoglubulin) antigen embedded in plastic: Prerequisites and limitations. J Histochem Cytochem 28: 1041–1049
Timpl R, Rohde H, Robey PG, Rennard SI, Foidart J-M, Martin GR (1979) Lamini — A glycoprotein from basement membranes. J Biol Chem 254: 9933–9937
Vuento M, Vaheri A (1979) Purification of fibronectin from human plasma by affinity chromatography under non-denaturing conditions. Biochem J 183: 331–337
Weber K, Bibring T, Osborn M (1975) Specific visualization of tubulin containing structures in tissue culture cells by immunofluorescence. Exp Cell Res 95: 111–120
Weber K, Pollack R, Bibring T (1975) Antibody against tubulin: the specific visualization of cytoplasmic microtubules in tissue culture cells. Proc Natl Acad Sci USA 72: 459–463
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
Weiss L, Greep RO (1977) Histology, 4th edn. McGraw-Hill, New York
Weingarten MD, Suter MM, Littman DR, Kirschner MW (1974) Properties of the depolymerization products of microtubules from mammalian brain. Biochemistry 13: 5529–5537
Witman GB, Carlson K, Rosenbaum JL (1972) Chlamydomonas flagella. II. The distribution of tubulins 1 and 2 in the outer doublet membrane. J Cell Biol 54: 540–555
Witman GB, Cleveland DW, Weingarten MD, Kirschner MW (1976) Tubulin requires tau for growth onto microtubule initiating sites. Proc Natl Acad Sci USA 73: 4070–4074
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Franklin, R.M., Martin, M.T. Pre-embedding immunohistochemistry as an approach to high resolution antigen localization at the light microscopic level. Histochemistry 72, 173–190 (1981). https://doi.org/10.1007/BF00517131
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00517131