The studies of mammalian brain are an intriguing part of molecular biology. The growing number of investigations of mechanisms implicated in brain development, function, and disease has brought new insights into our understanding of the mammalian nervous system organization. Pilot molecular cyto-genetic studies have indicated the occurrence of chromosomal variations manifested as aneuploidy in the developing and adult mammalian brain (1-4).
These findings lead to suggest that in coming years the molecular cytoge-netic analysis of brain tissues is going to be an important part of genome biology (4,5). The molecular cytogenetic analysis of different types of tissues often is complicated by misinterpretation of the results obtained. This is caused by in
From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.
Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ
large part neglecting tissue preparation specifications. Therefore, the detailed protocol of the most complicated tissues to prepare is of importance. The complicity of cultivating the majority of brain tissues has lead us to consider the interphase nuclei investigation of chromosome complement as a most appropriate way to conduct molecular cytogenetic analysis. To present the well-reproducing protocol of brain tissue processing for molecular cytogenetic analyses of chromosome complement in interphase nuclei, we have complied together the experience of different tissue preparations, such as developing brain, cultured fetal brain, frozen brain tissues, formalin-fixed brain tissue, and paraffin sections of brain tissues.
In addition to different brain tissue-processing protocols, we also have described the quality control procedure because it seems for us to be an important part of the successful molecular cytogenetic analysis. The quality control of the tissue preparation is the final step before the proceeding to primed in situ labeling (PRINS) (or fluorescence in situ hybridization) analysis. This procedure allows excluding the usefulness specimens not to make an unproductive use of molecular cytogenetic probes. There are two possibilities of control to be conducted. The visualization of suspension obtained by means of light microscope equipped by phase contrast optics provides by the information about the concentration and distribution of nuclei in cytogenetic slide preparation. In addition, the DAPI staining of nuclei allows assessing the quality of the suspension obtained and the level of autofluorescence by means of fluorescent microscope. However, it should be noted that light microscope visualization and DAPI staining are more effective when used together.
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