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Specificity controls for immunocytochemistry


Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.

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  1. Amari S, Beltrame F, Bjaalie JG, Dalkara T, De Schutter E, Egan GF, Goddard NH, Gonzalez C, Grillner S, Herz A, Hoffmann KP, Jaaskelainen I, Koslow SH, Lee SY, Matthiessen L, Miller PL, Da Silva FM, Novak M, Ravindranath V, Ritz R, Ruotsalainen U, Sebestra V, Subramaniam S, Tang Y, Toga AW, Usui S, Van Pelt J, Verschure P, Willshaw D, Wrobel A (2002) Neuroinformatics: the integration of shared databases and tools towards integrative neuroscience. J Integr Neurosci 1:117–128

  2. Beckstrøm H, Julsrud L, Haugeto Ø, Dewar D, Graham DI, Lehre KP, Storm-Mathisen J, Danbolt NC (1999) Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer’s disease. J Neurosci Res 55:218–229

  3. Bjørås M, Gjesdal O, Erickson JD, Torp R, Levy LM, Ottersen OP, Degree M, Storm-Mathisen J, Seeberg E, Danbolt NC (1996) Cloning and expression of a neuronal rat brain glutamate transporter. Mol Brain Res 36:163–168

  4. Burry RW (2000) Specificity controls for immunocytochemical methods. J Histochem Cytochem 48:163–166

  5. Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105

  6. Danbolt NC, Lehre KP, Dehnes Y, Chaudhry FA, Levy LM (1998) Localization of transporters using transporter-specific antibodies. Methods Enzymol 296:388–407

  7. Dehnes Y, Chaudhry FA, Ullensvang K, Lehre KP, Storm-Mathisen J, Danbolt NC (1998) The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J Neurosci 18:3606–3619

  8. Griffiths G. (1993) Fine structure of immunocytochemistry. Springer, Berlin Heidelberg New York

  9. Gundersen V, Danbolt NC, Ottersen OP, Storm-Mathisen J (1993) Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous D-aspartate. Neuroscience 57:97–111

  10. Habeeb AFSA, Hiramoto R (1968) Reaction of proteins with glutaraldehyde. Arch Biochem Biophys 126:16–26

  11. Hardy PM, Nicholls AC, Rydon HN (1976) The nature of the cross-linking of proteins by glutaraldehyde. Part 1. Interaction of glutaraldehyde with the amino-groups of 6-aminohexanoic acid and of alfa-N-acetyl-lysine. J Chem Soc Perkin Trans 9:958–962

  12. Haugeto Ø, Ullensvang K, Levy LM, Chaudhry FA, Honoré T, Nielsen M, Lehre KP, Danbolt NC (1996) Brain glutamate transporter proteins form homomultimers. J Biol Chem 271:27715–27722

  13. Holmseth S, Dehnes Y, Bjørnsen LP, Boulland JL, Furness DN, Bergles D, Danbolt NC (2005) Specificity of antibodies unexpected cross reactivity of antibodies directed against the EAAT3 (EAAC) glutamate transporter. Neuroscience 136:649–660

  14. Humbel BM, de Jong MD, Muller WH, Verkleij AJ (1998) Pre-embedding immunolabeling for electron microscopy: an evaluation of permeabilization methods and markers. Microsc Res Tech 42:43–58

  15. James JA, Mcclain MT, Koelsch G, Williams DG, Harley JB (1999) Side-chain specificities and molecular modelling of peptide determinants for two anti-Sm B/B′ autoantibodies. J Autoimmun 12:43–49

  16. Josephsen K, Smith CE, Nanci A (1999) Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin. J Histochem Cytochem 47:1237–1245

  17. Koslow SH, Subramaniam S (2005) Databasing the brain. From data to knowledge (neuroinformatics). Wiley, New York

  18. Lehre KP, Danbolt NC (1998) The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci 18:8751–8757

  19. Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC (1995) Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci 15:1835–1853

  20. Leong ASY, Gilham PN (1989) The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology (Phila) 21:266–268

  21. Levy LM, Lehre KP, Rolstad B, Danbolt NC (1993) A monoclonal antibody raised against an [Na+–K+]coupled L-glutamate transporter purified from rat brain confirms glial cell localization. FEBS Lett 317:79–84

  22. Levy LM, Lehre KP, Walaas SI, Storm-Mathisen J, Danbolt NC (1995) Down-regulation of glial glutamate transporters after glutamatergic denervation in the rat brain. Eur J Neurosci 7:2036–2041

  23. van Lookeren Campagne M, Oestreicher AB, van der Krift TP, Gispen WH, Verkleij AJ (1991) Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens. J Histochem Cytochem 39:1267–1279

  24. Mathiisen TM, Nagelhus EA, Jouleh B, Torp R, Frydenlunde DS, Mylonakou M, Amiry-Moghaddam M, Luciene C, Utvik JK, Riber B, Gujord KM, Knutsen J, Davanger S, Haug F-M, Rinvik E, Ottersen OP (2006) Postembedding immunogold cytochemistry of membrane molecules and amino acid transmitters in the central nervous system (in press)

  25. Ottersen OP (1989a) Postembedding immunogold labelling of fixed glutamate: an electron microscopic analysis of the relationship between gold particle density and antigen concentration. J Chem Neuroanat 2:57–66

  26. Ottersen OP (1989b) Quantitative electron microscopic immunocytochemistry of neuroactive amino acids. Anat Embryol (Berl) 180:1–15

  27. Petralia RS, Wenthold RJ (1999) Immunocytochemistry of NMDA receptors. In: Li M (ed) NMDA receptor protocols. Humana Press, Totowa, pp 73–92

  28. Pool CW, Buijs RM (1988) Antigen identity in immunocytochemistry. In: Van Leeuwen F, Buijs RM, Pool CW, Pach O (eds) Molecular neuroanatomy. Elsevier, Amsterdam, pp 233–266

  29. Roberg B, Torgner IA, Kvamme E (1995) The orientation of phosphate activated glutaminase in the inner mitochondrial membrane of synaptic and non-synaptic rat brain mitochondria. Neurochem Int 27(4–5):367–376

  30. Saper CB, Sawchenko PE (2003) Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. J Comp Neurol 465:161–163

  31. Sibille P, Ternynck T, Nato F, Buttin G, Strosberg D, Avrameas A (1997) Mimotopes of polyreactive anti-DNA antibodies identified using phage-display peptide libraries. Eur J Immunol 27:1221–1228

  32. Steyger PS, Furness DN, Hackney CM, Richardson GP (1989) Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure. Hear Res 42:1–16

  33. Storm-Mathisen J, Ottersen OP (1990) Immunocytochemistry of glutamate at the synaptic level. J Histochem Cytochem 38:1733–1743

  34. Storm-Mathisen J, Leknes AK, Bore AT, Vaaland JL, Edminson P, Haug F-MS, Ottersen OP (1983) First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301:517–520

  35. Swaab DF, Pool CW, Van Leeuwen FW (1977) Can specificity ever be proved in immunocytochemical staining. J Histochem Cytochem 25:388–391

  36. Tessler S, Danbolt NC, Faull RLM, Storm-Mathisen J, Emson PC (1999) Expression of the glutamate transporters in human temporal lobe epilepsy. Neuroscience 88:1083–1091

  37. Tijssen P, Burdon RH, van Knippenberg PH (1985) Practice and theory of enzyme immunoassays. Elsevier, Amsterdam

  38. Willingham MC (1999) Conditional epitopes. Is your antibody always specific? J Histochem Cytochem 47:1233–1236

  39. Zhang N, Storm-Mathisen J, Ottersen OP (1993) A model system for quantitation in single and double labelling postembedding electron microscopic immunocytochemistry. Elsevier Neuroscience Protocols 93-050-13:1–20

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We thank David N. Furness for discussions and critical reading of the manuscript, and Bjørg Roberg for the preparation of the purified mitochondria used in Fig. 1. This work was supported by the Norwegian Top Research Program (Toppforskningsprogrammet), the Norwegian Research Council (MH-group coordinated by Ottersen and fellowship to KPL), EU BIOMED (contract QLG3 CT 2001 02004).

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Correspondence to N. C. Danbolt.

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Holmseth, S., Lehre, K.P. & Danbolt, N.C. Specificity controls for immunocytochemistry. Anat Embryol 211, 257–266 (2006). https://doi.org/10.1007/s00429-005-0077-6

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  • Antibody specificity
  • Glutamate transporter
  • Proteomics
  • Antibody affinity