Takahashi R, Okuyama T, Matsuo S, Maeda S
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan.
Acta Cytol. 1996 May-Jun;40(3):396-400. doi: 10.1159/000333843.
To develop a new method of fixing and preserving cytologic specimens for immunostaining after long-term storage at room temperature.
The method consists of three steps: fixation, freeze substitution or freeze drying, and storage. To test the method, we used the human small cell lung carcinoma cell line Lu-135, which expresses a high level of mutant p53 protein and exhibits strong nuclear staining when reacted with an anti-p53 antibody. A smear of Lu-135 cells was fixed in a mixture of methanol and ether (50:50=vol/vol) or sprayed with a fixative containing isopropyl alcohol, methanol and polyethylene glycol. The fixed cells were freeze substituted by immersing them in a dry ice/methanol/ether bath and then were freeze dried under a vacuum. The smear was then placed in a 50-mL conical tube containing silica gel. The tube was sealed and stored at room temperature.
Freeze substituted cells that were fixed with methanol/ether and stored for more than six months retained strong p53 positivity, as strong as that of the control cells, which had been fixed and stored in methanol.
Freeze substitution and freeze drying are an alternative method of preserving cytologic specimens.
开发一种新方法，用于在室温下长期保存后对细胞学标本进行固定和保存，以便进行免疫染色。
该方法包括三个步骤：固定、冷冻置换或冷冻干燥以及储存。为了测试该方法，我们使用了人小细胞肺癌细胞系Lu-135，其表达高水平的突变型p53蛋白，并且在与抗p53抗体反应时呈现强烈的核染色。将Lu-135细胞涂片固定于甲醇和乙醚的混合物（50:50，体积/体积）中，或用含有异丙醇、甲醇和聚乙二醇的固定剂喷洒。将固定后的细胞浸入干冰/甲醇/乙醚浴中进行冷冻置换，然后在真空下进行冷冻干燥。然后将涂片置于含有硅胶的50 mL锥形管中。将管子密封并在室温下储存。
用甲醇/乙醚固定并储存超过6个月的冷冻置换细胞保留了强烈的p53阳性，与固定并储存在甲醇中的对照细胞一样强。
冷冻置换和冷冻干燥是保存细胞学标本的一种替代方法。
