Derick Mussen Preventive Healthcare

Basic Preparation Of Metaphase Chromosome Slides From Protocol 1 Peripheral Blood Lymphocytes

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Human metaphase chromosomes are prepared for FISH applications essentially according to routine cytogenetic procedures. The protocol described here consists of culturing whole blood, metaphase arrest with Colcemid (demecolcine), hypotonic treatment, fixation (Bosman et al., 1975), and slide preparation.

To increase accessibility of the target sequences for reaction with probe DNA and immunocytochemical reagents, chromosome preparations are pretreated prior to FISH with RNase A to remove any endogenous RNA that may be present. This is followed by protease treatment (e.g., with pepsin) to remove proteins, and finally a post-fixation treatment with formaldehyde to ensure preservation of chromosomal morphology (Wiegant et al., 1991).