INSITU HYBRIDIZATION HISTOCHEMISTRY - A NEW METHOD FOR PROCESSING MATERIAL STORED FOR SEVERAL YEARS
INSITU HYBRIDIZATION HISTOCHEMISTRY - A NEW METHOD FOR PROCESSING MATERIAL STORED FOR SEVERAL YEARS
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DOI：
10.1016/0006-8993(92)90243-3
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发表时间：
1992-04-24
期刊：
影响因子：
2.9
通讯作者：
HABER, SN
中科院分区：
文献类型：
--
作者：
LU, WX;HABER, SN
我们在这里描述了一种通过原位杂交检测特定 mRNA 的方案，使用未经灭活 RNase 处理并在冷冻保护剂溶液中保存数年的组织切片。将大鼠、猴子和人类的大脑切成 50 μm 的切片，并在 -20 摄氏度下自由漂浮于基于乙二醇的冷冻保护溶液中。大鼠脑切片在冷冻保护溶液中保存3天、1个月和2个月。切割对照切片并立即安装在涂有明胶的载玻片上并储存在-80℃下。猴脑切片在冷冻保护溶液中保存长达 5 年。人体切片在储存一年后进行测试。与人前脑啡肽原 mRNA（氨基酸序列 130-145）、大鼠前脑啡肽原 mRNA（序列 388-435）和大鼠酪氨酸羟化酶 mRNA（序列 1441-1488）互补的寡核苷酸探针用 S-35-dATP 和末端脱氧核苷酸转移酶标记。为了防止将组织切片安装到明胶包被的载玻片上可能出现的 RNase 污染，原位杂交在无菌培养皿中进行。每一步之后，将溶液从培养皿中吸出。每个切片所需的探针量为45μl（大鼠）、450μl（猴）和350μl（人）。使用该方案，与在明胶包被的侧面上后处理的对照切片相比，大鼠脑切片中特定 mRNA 的检测更具特异性，非特异性背景更少。在冷冻保护剂中保存长达 31 个月的猴脑切片和保存 12 个月的人脑切片也获得了出色的分辨率。与传统的原位杂交方法类似，不需要预杂交处理步骤来降低背景。使用该方案，可以在冷冻保护剂溶液中保存数年的脑切片中获得低背景的特定 mRNA 标记。
We describe here a protocol developed to detect specific mRNAs by in situ hybridization using tissue sections that were not treated to inactivate RNase and were stored in cryoprotectant solution for several years. Brains from rats, monkeys and humans were sectioned at 50 mu-m and stored free floating in an ethylene glycol based cryoprotective solution at -20-degrees-C. Rat brain sections were kept in cryoprotective solution for 3 days, 1 month and 2 months. Control sections were cut and mounted immediately on gelatin-coated slides and stored at -80-degrees-C. Monkey brain sections were stored in cryoprotective solution for up to 5 years. Human sections were tested after storage for one year. Oligonucleotide probes that were complementary to human preproenkephalin mRNA (amino acid sequences 130-145), rat preproenkephalin mRNA (sequences 388-435) and rat tyrosine hydroxylase mRNA (sequences 1441-1488) were labeled with S-35-dATP and terminal deoxynucleotidyl transferase. To prevent possible RNase contamination from mounting the tissue sections onto gelatin coated slides, the in situ hybridization was performed in sterile culture dishes. Following each step, solutions were aspirated out of the dish. The amount of probe necessary for each section was 45-mu-l (rat), 450-mu-l (monkey), and 350-mu-l (human). Using this protocol, the detection of specific mRNAs in rat brain sections was more specific with less non-specific background as compared to control sections that were processed after they were mounted onto gelatin-coated sides. Excellent resolution was also obtained from monkey brain sections that were stored in cryoprotectant for up to 31 months and in human brain sections stored for 12 months. Similar to conventional in situ hybridization methodology, the prehybridization treatment step was not necessary to reduce the background. Using this protocol, it is possible to obtain specific mRNA labeling with low background in brain sections that have been stored for several years in cryoprotectant solution.