| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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Male adult Sprague-Dawley rats were used for in situ hybridization. The frozen tissues were sectioned with Microm HM 500 cryostat at 14 µm and thaw-mounted onto polysine glass slides (Menzel, Germany). The oligonucleotides used in hybridization corresponded to nucleotides 1474-1518 (antisense) of rat ODC mRNA (Wen et al. 1989) and to nucleotides 71-109 (antisense) of rat antizyme mRNA (Matsufuji et al. 1995) and were labeled at the 3’end with [33P]dATP (New England Nuclear Research Products, Boston, MA, USA). Several control probes with the same length and similar GC content and specific activity were used to determine the specificity of the hybridization. The specificity of probes was also verified by Northern analysis.
In situ hybridization was carried out as described previously (Kononen & Pelto- Huikko 1997). After hybrization the sections were either covered with Kodak Biomax MR autoradiofilm (Kodak, Rochester, NY, USA) for 30-60 days or dipped in Kodak NTB2 nuclear track emulsion and exposed 90 days at 4¢ªC. The hybridization signals were quantified using an image analysis system consisting of IBM-PC, Sensi-Cam digital camera (PCO Computer Optics GmbH, Kelheim, Germany), Nikon 55-mm lens and Northern Light precision illuminator (Imaging Research, St. Catharine’s, Ontario, Canada). The measurements were carried out using Image-Pro Plus programme (Media Cybernetics, Silver Spring, MD, USA). The grey levels corresponding to 14C-plastic standards (Amersham International, Buckingham-shire, UK) lying within the exposure range of the film were determined and used in a Lagrange approximation to construct the grey level to activity transfer function. The borders of the measuring fields were interactively defined, and the average activity in the tissue fields was calculated. At least five sections were measured, and the mean value was used.