This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Rieux, C. Articles by Cooper, H. M. Search for Related Content PubMed PubMed Citation Articles by Rieux, C. Articles by Cooper, H. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB AGAR-AGAR BIOTIN Social Bookmarking                         What's this?

Analysis of Immunohistochemical Label of Fos Protein in the Suprachiasmatic Nucleus: Comparison of Different Methods of Quantification

Institut National de la Santé de la Recherche Médicale Unité 371, Cerveau et Vision, 18 Avenue du Doyen Lépine, 69675 Bron, France

R. Carney

D. Lupi

Institut National de la Santé de la Recherche Médicale Unité 371, Cerveau et Vision, 18 Avenue du Doyen Lépine, 69675 Bron, France; Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Campus, St. Dunstan's Road, London W6 8RF, United Kingdom

O. Dkhissi-Benyahya

Institut National de la Santé de la Recherche Médicale Unité 371, Cerveau et Vision, 18 Avenue du Doyen Lépine, 69675 Bron, France

K. Jansen

Biological Center, University of Groningen, Kerklaan 30, P.O. Box 14, 9750 AA Haren, the Netherlands; Tissue Engineering/Medical Biology, Academic Hospital—University of Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands.

N. Chounlamountri

Institut National de la Santé de la Recherche Médicale Unité 371, Cerveau et Vision, 18 Avenue du Doyen Lépine, 69675 Bron, France

R. G. Foster

Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Campus, St. Dunstan's Road, London W6 8RF, United Kingdom

H. M. Cooper

Institut National de la Santé de la Recherche Médicale Unité 371, Cerveau et Vision, 18 Avenue du Doyen Lépine, 69675 Bron, France; cooper{at}lyon151.inserm.fr

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.

Key Words: circadian rhythm • image analysis • optical density • photoreception • stereology • cell counts • immediate early genes

Journal of Biological Rhythms, Vol. 17, No. 2, 121-136 (2002)
DOI: 10.1177/074873002129002410


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				L. Muscat and L. P. Morin Binocular Contributions to the Responsiveness and Integrative Capacity of the Circadian Rhythm System to Light J Biol Rhythms, December 1, 2005; 20(6): 513 - 525. [Abstract] [PDF]