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Indirect immunofluorescence of unfixed mammalian metaphase chromosomes: method and applications^*

Peter Jeppesen

MRC Human Genetics Unit, Western Genera1 Hospital, Crewe Road, Edinburgh EH4 2XU, UK

ABSTRACT

Conventional fixation for preparing mammalian metaphase chromosome spreads, for example, the use of methanol:acetic acid (3:1), is not, in general, suitable for immunofluorescence studies. Basic proteins, such as histones, are extracted wider acidic conditions, and other chromosomal proteins ofter stiffer reduced or tota1 loss of anligenicily following acid denaturation. To avoid these problems, a method of preparing metaphase spreads by cytocentrifugation has been developed that avoids altogether the need for prior fixation, thus ensuring maximum retention of in vivo chromosome conformation, and unimpaired antigenicity of chromosoma1 components during reaction with antibodies. The method is presented and illustrated with examples of immunofluorescent labelling of metaphases from a number of species, using antoimmune sera and antibodies to defined histone epitopes. An extension of the method in double-labelling type experiments to localize one antigen with respect to another, or an antigen with respect to DNA sequence, is also demonstrated.

Keywords: Immunofluorescence; Mammalian metaphase chromosomes.

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* This paper was presented at the ICRO/UNESCO International Training Course "Molecular Organization of the Eukaryotic Chromosome. in Relation to the Induction of Chromosome Aberrations" organized by Professor Gunter Obe, Dr. Máximo E. Drets and Dr. Gustavo A. Folle, held September 16-28, 1991 at Montevideo, Uruguay. It is the second in a series of five papers from this course which will be published in the Brazilian Journal of Genetics.