KOSEN 한인과학기술자네트워크

>neurodegeneration을 알아보기위해 뇌조직에서 >Amino cupric silver staining 실험을 하려고 합니다. >실험법에 대해 알고 계신 내용이나 >자료를 알려주세요... A serial set of every sixth section (a 240 µm interval) was selected for staining with the amino-cupric-silver stain of de Olmos (1994) to reveal disintegrative degeneration. The free-floating sections were taken through the following major steps: preimpregnation, impregnation, reduction, bleaching, and fixing. The preimpregnation solution contained cupric nitrate, silver nitrate, cadmium nitrate, lanthanum nitrate, neutral red, -amino butyric acid, alanine, pyridine, triethanolamine, isopropanol, and deionized water. After the components were well mixed, the solution was microwaved until it reached 45-50°C. The solution was left to cool to room temperature, then filtered. The sections were removed from the cacodylate-buffered formaldehyde and rinsed with deionized water. They were then placed into dishes containing the preimpregnation solution and heated in the microwave to 45-50°C. To allow for cooling, the sections remained in this solution overnight. The impregnation solution contained silver nitrate, 100% ethanol, acetone, lithium hydroxide, ammonium hydroxide, and deionized water. The sections were rinsed first in deionized water, second in acetone, and then placed into the impregnation solution. They incubated in this solution for 50 min. The reducer solution contained 100% ethanol, formalin, citric acid, and deionized water. The sections were transferred from the impregnation solution into the reducer solution and placed in a water bath with a maintained temperature between 32 and 35°C. After 25 min in the reducer solution, the sections were transferred into deionized water rinses, then an acetic acid rinse and back into deionized water. The bleaching solutions were potassium ferricyanide in potassium chlorate with lactic acid, potassium permanganate with sulfuric acid, and sodium thiosulfate. The sections were rapidly transferred through these bleaching solutions, then fixed in rapid fixer solution for 1 min 30 sec. The sections were then rinsed in deionized water, mounted on subbed glass slides, and counterstained with neutral red to reveal normal cell bodies.