Tissue Fixation Prevents Contamination of Tritium-Sensitive Storage Phosphor Imaging Plates

The use of storage phosphor imaging plates is becoming an increasingly popular alternative to X-ray film when detection speed rather than spatial resolution is the prime concern for detection of radioactive decay during in situ hybridization, northern/Southern blotting and radioligand binding assays. Because the photo-stimulable imaging plates are approximately 5 times more sensitive than traditional film, exposure times can be dramatically shortened (2). The imaging plates are placed in contact with the sample in exactly the same way as film (but without the need for darkroom conditions), and an image is captured in the coating of fine crystals of halogenated barium phosphors containing traces of Europium rather than the silver halides of X-ray film. The latent image is retrieved by instruments such as the Fuji BAS 3000 (Fuji Photo Film Ltd., Tokyo, Japan), which uses a photomultiplier to measure the light emitted from an irradiated plate as an He/Ne laser scans across its surface. Imaging plates come in two formats. The standard plates, used to detect most radiolabels, are covered by a robust protective coating and are thus readily reusable, keeping costs low. However, to detect tritiated probes, plates without the protective coating must be used to allow the low-energy radiation to reach the detecting phosphors. This presents a problem for radioligand binding assays (4,5) to detect cell-surface receptors. Because these assays are generally performed on unfixed tissues, and the interaction of the ligand with its receptor is relatively weak (in a chemical sense), a proportion of the ligand can diffuse away from the receptor and permanently contaminate the imaging plate. This phenomenon is not seen with all ligands. For example, in our hands, the dopamine D2 ligand [3H]-sulpiride (3) does not cause contamination, while the dopamine D1 ligand [3H]-SCH-23390 (1) does cause contamination. To try to prevent this contamination and allow repeated use of the tritiumsensitive imaging plates, we have examined the ability of fixation by immersion in 10% formalin, 4% paraformaldehyde, 3% gluteraldehyde or vapor fixation in paraformaldehyde, to fix the radioligand [3H]-SCH-23390 into coronal sections of mouse brain after binding to striatal dopamine D1 receptors. Tissue for this experiment was obtained from five male C-57 Black/6J mice (17–25 g and 5–6 weeks of age) that were killed by nembutal overdose (600 mg/kg, i.p.). Then, the brains were removed and snap-frozen in dry icecooled isopentane for storage at -80°C. Twenty-micron serial coronal sections were cut through the striatum using a Model 5030 Cryostat (Bright Instruments, Huntingdon, Cambs, England, UK), with the chamber and blade maintained at -20°C and thaw-mounted onto gelatin-chrome, alum-coated glass slides, which were then desiccated and stored at -80°C until needed for the assay. Dopamine D1 receptor density was measured using the method of Cortes et al. (1). Briefly, sections were washed at room temperature in 50 mM Tris-HCl buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM mianserin, pH 7.4). The mianserin was added to prevent binding to serotonin receptors. Then, the sections were incubated for 1 h at room temperature in the same buffer containing 1 nM [3H]SCH-23390 (NEN Life Science Products, Boston, MA, USA) with a specific activity of 85.5 Ci/mMol. After incubation, the sections were prewashed by quickly dipping in 50 mM Tris-HCl buffer at 0°C and then washed twice for 10 min in the same buffer at 0°C. Then the sections were rinsed in ice-cold distilled water for 5 s and dried under a stream of cold air before vacuum desiccation. Nonspecific binding was determined on alternative sections that were incubated as above but with an extra 1 nM unlabeled SCH-23390 added to the incubation medium. After the assay, the slides were sequentially divided into five series, each providing a representative sample of the striatum from each mouse. Sections from the control (unfixed) series were stored overnight under vacuum at 4°C. For fixation, three of the other series of slides were incubated overnight at 4°C in 10% formalin, 4% paraformaldehyde or 3% gluteraldehyde, respectively, dissolved in phosphate-buffered saline (PBS), pH 7.4. The fifth series of slides were fixed by placing the slides in a