Summary
Repeated application of the first-layer antiserum in the indirect immunofluorescence technique considerably improves the immunostaining. The modified method reveals more antigenic sites and increases the contrast between specific and background stainings, particularly where sparsely distributed antigenic areas are to be investigated. The effect of this novel immunostaining procedure is compared with that of the routine procedure and of other modifications. Possible mechanisms for the improving effect are discussed. A procedure of combined modifications is recommended for exploration of non-abundant antigens or for achieving a high-quality photograph.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bishop, A. E., Polak, J. M., Bloom, S. R. &Pearse, A. G. E. (1978) A new universal technique for the immunocytochemical localisation of peptidergic innervation.J. Endocrinol. 77, 25–6.
Brandtzaeg, P. (1981) Prolonged incubation staining of immunoglobulins and epithelial components in ethanol- and formaldehyde-fixed paraffin-embedded tissues.J. Histochem. Cytochem. 29, 1302–15.
Coons, A. H., Leduc, E. H. &Connolly, J. M. (1955) Studies on antibody production.J. exp. Med. 102, 49–60.
Dorsett, B. H. &Joachim, H. L. (1978) A method for the use of immunofluorescence on paraffin-embedded tissues.Am. J. clin. Path. 69, 66–72.
Goldstein, M. (1972) Enzymes involved in the catalysis of catecholamine biosynthesis. InMethods in Neurochemistry (edited byRodnight, R. andMarke, N.), pp. 317–340. New York: Plenum Press.
Gu, J., De Mey, J., Moeremans, M. &Polak, J. M. (1981) Sequential use of the PAP and immunogold staining methods for the light microscopical double staining of tissue antigens, its application to the study of regulatory peptides in the gut.Regulatory Peptides 1, 365–74.
Hartman, B. K. (1973) Immunofluorescence of dopamine-β-hydroxylase. Application of improved methodology to the localization of the peripheral and central noradrenergic nervous system.J. Histochem. Cytochem. 21, 312–32.
Hartman, B. K., Zide, D. &Adenfriend, S. (1972) The use of dopamine hydroxylase as a marker for the central noradrenergic nervous system in the rat brain.Proc. natn. Acad. Sci., U. S. A. 69, 2722–6.
Huang, S. N., Minassian, H. &More, J. D. (1976) Application of immunofluorescent staining on paraffin sections improved by trypsin digestion.Lab. Invest. 35, 383–90.
Marangos, P. J., Schmechel, D. E., Parma, A., Clark, R. L. &Goodwin, F. K. (1979) Measurement of neuron-specific (NSE) and non-neuronal (NNE) isoenzymes of enolase in rat, monkey and human nervous tissue.J. Neurochem. 33, 319–29.
Pearse, A. G. E. &Polak, J. M. (1975) Bifunctional reagents as vapour and liquid phase fixatives for immunocytochemistry.Histochem. J. 7, 179–86.
Polak, J. M., Gu, J., Mina, S. &Bloom, S. R. (1981) VIPergic nerves in the penis.Lancet 2, 217–9.
Portman, B., Galbraith, R. M., Eddleston, A. L. W. F., Zuckerman, A. J. &Williams, R. (1976) Detection of HBsAg in fixed liver tissue-use of a modified immunofluorescence technique and comparison of histochemical methods.Gut 17, 1–6.
Scott, H., Brandtzaeg, P., Solheim, B. G. &Thorsby, E. (1981) HLA-DR-like antigens and secretory component (SC) in jejunal epithelium of patients with coeliac disease or dermatitis herpetiformis.Clin. exp. Immun. 44, 233–8.
Sternberger, L. A. (1974) The unlabelled antibody enzyme method. InImmunocytochemistry. pp. 129–171. New Jersey: Prentice-Hall.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gu, J., Islam, K.N. & Polak, J.M. Repeated application of first-layer antiserum improves immunofluorescence staining: a modification of the indirect immunofluorescence staining procedure. Histochem J 15, 475–482 (1983). https://doi.org/10.1007/BF01002701
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01002701