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Whole-mount Immunohistochemistry: A High-throughput Screen for Patterning Defects in the Mouse Cerebellum
Roy V. Sillitoea and Richard Hawkesaa Department of Cell Biology & Anatomy, and Genes and Development Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
Correspondence to: Richard Hawkes, Dept. of Cell Biology & Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada. E-mail: rhawkes@ucalgary.ca
Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.
(J Histochem Cytochem 50:235–244, 2002)
Key Words: Purkinje cell, zebrin, mutagenesis, calbindin, lurcher, NPC, pattern formation
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