|
|
|
|
 |
|||
|
|||
A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology
HY Lan, W Mu, YY NG, DJ Nikolic-Paterson and RC Atkins
Department of Nephrology, Monash Medical Centre, Clayton, Victoria, Australia.
The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.
Volume 44,
Issue 3,
pp. 281-287,
03/01/1996
Copyright © 1996 by The Histochemical Society
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Shimazaki, Y. Kawamura, A. Kanazawa, A. Sekine, S. Saito, T. Tsunoda, D. Koya, T. Babazono, Y. Tanaka, M. Matsuda, et al. Genetic Variations in the Gene Encoding ELMO1 Are Associated With Susceptibility to Diabetic Nephropathy Diabetes, April 1, 2005; 54(4): 1171 - 1178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Deininger, B. A. Fimmen, D. R. Thal, H. J. Schluesener, and R. Meyermann Aberrant Neuronal and Paracellular Deposition of Endostatin in Brains of Patients with Alzheimer's Disease J. Neurosci., December 15, 2002; 22(24): 10621 - 10626. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Mitchell, K. Feyereisen, S. Bouret, D. Leroy, and J.-C. Beauvillain Microwave Strategy for Improving the Simultaneous Detection of Estrogen Receptor and Galanin Receptor mRNA in the Rat Hypothalamus J. Histochem. Cytochem., July 1, 2001; 49(7): 901 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Elmlinger, M. H. Deininger, B. S. Schuett, R. Meyermann, F. Duffner, E. H. Grote, and M. B. Ranke In Vivo Expression of Insulin-Like Growth Factor-Binding Protein-2 in Human Gliomas Increases with the Tumor Grade Endocrinology, April 1, 2001; 142(4): 1652 - 1658. [Abstract] [Full Text]
|
|
|
|
 |
|
 S.-G. Lin, X.-Y. Yu, Y.-X. Chen, X. R. Huang, C. Metz, R. Bucala, C.-P. Lau, and H. Y. Lan De Novo Expression of Macrophage Migration Inhibitory Factor in Atherogenesis in Rabbits Circ. Res., December 8, 2000; 87(12): 1202 - 1208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kitayama, H. Igarashi, and H. Sugimura Different Vulnerability among Chromosomes to Numerical Instability in Gastric Carcinogenesis: Stage-dependent Analysis by FISH with the Use of Microwave Irradiation Clin. Cancer Res., August 1, 2000; 6(8): 3139 - 3146. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. M. Shelton, M.-H. Lee, J. A. Richardson, and S. B. Patel Microsomal triglyceride transfer protein expression during mouse development J. Lipid Res., March 1, 2000; 41(4): 532 - 537. [Abstract] [Full Text]
|
|
|
|
|
|
F.-J. Lai, S.-J. Shin, Y.-J. Lee, S.-R. Lin, W.-Y. Jou, and J.-H. Tsai Up-Regulation of Adrenal Cortical and Medullary Atrial Natriuretic Peptide and Gene Expression in Rats with Deoxycorticosterone Acetate-Salt Treatment Endocrinology, January 1, 2000; 141(1): 325 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-Y. Ng, J.-M. Fan, W. Mu, D. J. Nikolic-Paterson, W.-C. Yang, T.-p. Huang, R. C. Atkins, and H. Y. Lan Glomerular epithelial-myofibroblast transdifferentiation in the evolution of glomerular crescent formation Nephrol. Dial. Transplant., December 1, 1999; 14(12): 2860 - 2872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Oliver, R. P. Heavens, and D. J. S. Sirinathsinghji Quantitative Comparison of Pretreatment Regimens Used to Sensitize In Situ Hybridization Using Oligonucleotide Probes on Paraffin-embedded Brain Tissue J. Histochem. Cytochem., December 1, 1997; 45(12): 1707 - 1714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Y. Lan, M. Bacher, N. Yang, W. Mu, D. J. Nikolic-Paterson, C. Metz, A. Meinhardt, R. Bucala, and R. C. Atkins The Pathogenic Role of Macrophage Migration Inhibitory Factor in Immunologically Induced Kidney Disease in the Rat J. Exp. Med., April 21, 1997; 185(8): 1455 - 1466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. L. Eliasson, S. Blackshaw, M. J. Schell, and S. H. Snyder Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain PNAS, April 1, 1997; 94(7): 3396 - 3401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-R. Shi, R. J. Cote, and C. R. Taylor Antigen Retrieval Immunohistochemistry: Past, Present, and Future J. Histochem. Cytochem., March 1, 1997; 45(3): 327 - 344. [Abstract] [Full Text] [PDF]
|
|
| Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact |