|
|
|
|
 |
|||
|
|||
Optimization of immunofluorescence methods by quantitative image analysis
DE Mosedale, JC Metcalfe and DJ Grainger
Department of Biochemistry, University of Cambridge, United Kingdom.
There is a growing trend towards the objective quantification of immunohistochemical staining. However, quantification has not been used previously to optimize the original published immunohistochemical methods. We present a quantitative method for analyzing immunofluorescence staining employing the Applied Imaging MAGISCAN image analysis system, which has then been used to optimize major aspects of the standard immunofluorescent staining protocols. The optimization process resulted in a method that increased specific staining up to fivefold over typical published protocols, with no increase in nonspecific staining. The method is extremely reproducible. For slides stained by a single experimenter in one batch on one day, the coefficient of variation between replicate means is 1.2%. The image analysis protocol gave a linear response with increasing antigen concentration, as determined by using purified antigen dried onto slides. The revisions to the standard protocol presented here can also be applied to nonquantitative staining. It will help users of immunofluorescence to maximize their staining and may enable the detection of previously undetected antigens.
Volume 44,
Issue 9,
pp. 1043-1050,
09/01/1996
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:
|
|
 |
|
  D. J. Grainger, J. Reckless, and E. McKilligin Apolipoprotein E Modulates Clearance of Apoptotic Bodies In Vitro and In Vivo, Resulting in a Systemic Proinflammatory State in Apolipoprotein E-Deficient Mice J. Immunol., November 15, 2004; 173(10): 6366 - 6375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Johansson, E. Visse, B. Widegren, H.-O. Sjogren, and P. Siesjo Computerized Image Analysis as a Tool to Quantify Infiltrating Leukocytes: A Comparison Between High- and Low-magnification Images J. Histochem. Cytochem., September 1, 2001; 49(9): 1073 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Matkowskyj, D. Schonfeld, and R. V. Benya Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Using Commercially Available Software Photoshop and Matlab J. Histochem. Cytochem., February 1, 2000; 48(2): 303 - 312. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. Grainger, D. Mosedale, J. Metcalfe, and E. Bottinger Dietary fat and reduced levels of TGFbeta1 act synergistically to promote activation of the vascular endothelium and formation of lipid lesions J. Cell Sci., January 7, 2000; 113(13): 2355 - 2361. [Abstract] [PDF]
|
|
|
|
 |
|
 J. Reckless, E. M. Rubin, J. B. Verstuyft, J. C. Metcalfe, and D. J. Grainger Monocyte Chemoattractant Protein-1 but Not Tumor Necrosis Factor-{alpha} Is Correlated With Monocyte Infiltration in Mouse Lipid Lesions Circulation, May 4, 1999; 99(17): 2310 - 2316. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-A. Lehr, C. M. van der Loos, P. Teeling, and A. M. Gown Complete Chromogen Separation and Analysis in Double Immunohistochemical Stains Using Photoshop-based Image Analysis J. Histochem. Cytochem., January 1, 1999; 47(1): 119 - 126. [Abstract] [Full Text]
|
|
|
|
|
|
D. Grainger, J. Metcalfe, A. Grace, and D. Mosedale Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo J. Cell Sci., January 10, 1998; 111(19): 2977 - 2988. [Abstract] [PDF]
|
|
|
|
 |
|
 R. M. Lawn, A. D. Pearle, L. L. Kunz, E. M. Rubin, J. Reckless, J. C. Metcalfe, and D. J. Grainger Feedback Mechanism of Focal Vascular Lesion Formation in Transgenic Apolipoprotein(a) Mice J. Biol. Chem., December 6, 1996; 271(49): 31367 - 31371. [Abstract] [Full Text] [PDF]
|
|
| Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact |