Abstract
During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IκBα, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe–eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Al-Shamkhani A, Mallett S, Brown MH, James W, Barclay AN (1997) Affinity and kinetics for the interaction between soluble trimericOX40 ligand, a member of the tumour necrosis factor superfamily, and its receptor OX40 on activated T cells. J Biol Chem 272: 5275–5282.
Alam SM, Travers PJ, Wung JL, Nasholds W, Redpath S, Jameson SC, Gascoigne NR (1996) T-cell-receptor affinity and thymocyte positive selection. Nature 381: 616–620.
Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP (1993) Expression of human mucin genes in respiratory, digestive and reproductive tracts ascertained by in situ hybridisation. J Histochem Cytochem 41: 1479–1485.
Barrett DM, Faigel DO, Metz DC, Montone K, Furth EE (1997). In situ hybrdisation for Helicobacter pylori in gastric mucosal biopsy specimens: Quantitative evaluation of test performances in comparison with the CLO test and thiazine stain. J Clin Laborat Anal 11: 374–379.
Beckett CG, Dell'olio D, Ellis HJ, Rosen-Bronsen S, Ciclitira PJ (1998) The detection and localisation of inducible nitric oxide synthase production in the small intestine of patients with coeliac disease. Eur J Gastroenterol Hepatol 10: 641–647.
Cappello M, Keshav S, Prince C, Jewell DP, Gordon S (1992) Detection of mRNAs for macrophage products in inflammatory bowel disease by in situ hybridisation. Gut 33: 1214–1219.
Desreumaux P, Janin A, Delaporte E, Dubucquoi S, Piette F, Cortot A, Capron M, Colombel JF (1995) Parallel interleukin 5 synthesis by eosinophils in duodenal and skin lesion of a patient with dermatitis herpatiformis. Gut 37: 132–135.
Dubucquoi S, Janin A, Klein O, Desreumaux P, Quandalle P, Cortot A, Capron M, Colombel JF (1995) Activated eosinophils and interleukin 5 expression in early recurrence of Crohn's disease. Gut 37: 242–246.
Ellis R, Goodlad JR, Limb GA, Powell JP, Thompson RPH, Punchard NA (1998) Activation of NFκB in Crohn's Disease. Inflamm Res 47: 440–445.
Fox CH, Kotler D, Tierney A,Wilson CS, Fauci AS (1989) Detection of HIV-1 RNA in the lamina propria of patients with AIDS and gastrointestinal disease. J Infect Dis 159: 467–471.
Freeman TC, Howard A, Bentsen TS, Legon S, Walters JR (1995) Cellular and regional expression of transcripts of the plasma membrane calcium pump PMCA1 in rabbit intestine. Am J Physiol 269: 126–131.
Goldberg PA, Herbst F, Beckett CG, Martelli B, Kontakou M, Talbot IC, Ciclitira PJ, Nicholls RJ (1996) Leucocyte typing, cytokine expression, and epithelial turnover in the ileal pouch in patients with ulcerative colitis and familial adenomatous polyposis. Gut 38: 549–553.
Hamid Q, Azzawi M, Ying S, Moqbel R, Wardlaw AJ, Corrigan CJ, Bradley B, Durham SR, Collins JV, Jeffery PK, Quint DJ, Kay AB (1991) Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J Clin Invest 87: 1541–1546.
Haskill S, Beg AA, Tompkins S, Morris JS, Yurochko AD, Sampson-Johannes A, Mondal K, Ralph P, Baldwin AS (1991) Characterization of an immediate-early gene induced in adherent monocytes that encodes IkB-like activity. Cell 65: 1281–1289.
Huber CG, Oefner PJ, Guenther KB (1993) High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrenedivinylbenzene copolymers. Anal Biochem 212: 351–358.
Jonssen U, Fagerstam L, Roos H, Ronnberg J, Sjolander S, Sternberg E, Stahlberg R, Urbaniczky C, Ostlin H, Malmqvist M (1991) Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Bio Techniques 11: 620–627.
Kontakou M, Przemioslo RT, Sturgess RP, Limb GA, Ellis HJ, Day P, Ciclitira PJ (1995) Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge. Gut 37: 52–57.
Limb GA, Early O, Jones SE, Leroy F, Chignell AH, Dumonde DC (1994) Expression of mRNA coding for TNFα, IL-1β and IL-6 by cells infiltrating retinal membranes. Graefes Arch Clin Exp Ophthalmol 232: 646–651.
Lofas S, Johnsson B (1990) A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilisation of ligands. J Chem Soc 21: 1526–1528.
Mezey E, Hoffman BJ, Harta G, Palkovits M, Northup J (1994) Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune system. J Histochem Cytochem 42: 1277–1283.
Mullin GE, Maycon ZR, Braun-Elwert L, Cerchia R, James SP, Katz S, Weissman GS, Mckinley MJ, Fisher SE (1996) Inflammatory bowel disease mucosal biopsies have specialised lymphokine mRNA profiles. Inflamm Bowel Dis 2: 16–26.
Neurath MF, Becker C, Barbulescu K (1998) Role of NFB in immune and inflammatory responses in the gut. Gut 43: 856–860.
Ohno I, Lea RG, Flanders KC, Clark DA, Banwatt D, Dolovich J, Denburg J, Harley CB, Gauldie J, Jordana M (1992) Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor beta 1 gene. J Clin Invest 89: 1662–1668.
Patterson S, Gross J, Webster AD (1989) DNA probes bind nonspecifically to eosinophils during in situ hybridisation: Carbol chromotrope blocks binding to eosinophils but does not inhibit hybridisation to specific nucleotide sequences. J Virol Meth 23: 105–109.
Peterson CGB, Jornvall H, Venge P (1988) Purification and characterisation of eosinophil cationic protein from normal human eosinophils. Eur J Haematol 40: 415–423.
Pringle JH, Primrose L, Kind CN, Talbot IC, Lauder I (1989). In-situ hybridisation demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly-d(T) probe. J Pathol 158: 279–286.
Spry CJF (1996) Eosinophils. A Comprehensive Review & Guide to the Scientific & Medical Literature. Oxford: Oxford University Press, pp. 30–64.
Stahl LW, Eakin TJ, Baskin DG (1993) Selection of oligonucleotide probes for detection of mRNA isoforms. J Histochem Cytochem 41: 1735–1740.
Wantzin GL, Philip P, Killmann SA (1979) Cytoplasmic labelling of eosinophils with tritiated thymidine triphosphate. Biomedicine 31: 10–11.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ellis, R.D., Ashwood, P., Powell, J.J. et al. Selective Binding of Nucleotide Probes by Eosinophilic Cationic Protein During in situ Hybridisation. Histochem J 34, 153–160 (2002). https://doi.org/10.1023/A:1020942531028
Issue Date:
DOI: https://doi.org/10.1023/A:1020942531028