INDIRECT IMMUNOFLUORESCENCE OF UNFIXED MAMMALIAN METAPHASE CHROMOSOMES - METHOD AND APPLICATIONS

Conventional fixation for preparing mammalian metaphase chromosome spr eads, for example, the use of methanol:acetic acid (3:1), is not, in g eneral, suitable for immunofluorescence studies. Basic proteins, such as histones, are extracted under acidic conditions, and other chromoso mal proteins offer suffer reduced or total loss of antigenicity follow ing 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 retenti on of in vivo chromosome conformation, and unimpaired antigenicity of chromosomal components during reaction with antibodies. The method is presented and illustrated with examples of immunofluorescent labelling of metaphases from a number of species, using autoimmune sera and ant ibodies to defined histone epitopes. An extension of the method in dou ble-labelling type experiments to localize one antigen with respect to another, or an antigen with respect to DNA sequence, is also demonstr ated.