Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
Am J Physiol Lung Cell Mol Physiol. 2014 Feb 15;306(4):L341-50. doi: 10.1152/ajplung.00329.2013. Epub 2013 Dec 27.
Preservation of original tissue dimensions is an essential prerequisite for morphometric studies. Shrinkage occurring during tissue processing for histology may severely influence the appearance of structures seen under the microscope and stereological calculations. Therefore, shrinkage has to be avoided so that estimates obtained by application of unbiased stereology are indeed unbiased. The present study investigates the alterations of tissue dimensions of mouse lung samples during processing for histology. Different fixatives as well as embedding protocols are considered. Mouse lungs were fixed by instillation of either 4% formalin or a mixture of 1.5% glutaraldehyde/1.5% formaldehyde. Tissue blocks were sampled according to principles of stereology for embedding in paraffin, glycol methacrylate without treatment with osmium tetroxide and uranyl acetate, and glycol methacrylate including treatment with osmium tetroxide and uranyl acetate before dehydration. Shrinkage was investigated by stereological measurements of dimensional changes of tissue cut faces. Results show a shrinkage of the cut face areas of roughly 40% per lung during paraffin embedding, 30% during "simple" glycol methacrylate embedding, and <3% during osmium tetroxide/uranyl acetate/glycol methacrylate embedding. Furthermore, the superiority of the glutaraldehyde-containing fixative regarding shrinkage is demonstrated. In conclusion, the use of a glutaraldehyde-containing fixative and embedding in glycol methacrylate with previous treatment of the samples with osmium tetroxide and uranyl acetate before dehydration is recommended for stereological studies of the mouse lung.
保持组织原有尺寸是形态计量学研究的基本前提。组织在进行组织学处理过程中发生的收缩可能会严重影响显微镜下观察到的结构和体视学计算的结果。因此，必须避免收缩，以便通过应用无偏体视学获得的估计值确实是无偏的。本研究调查了在进行组织学处理过程中鼠标肺样本的组织尺寸变化。考虑了不同的固定剂和包埋方案。通过向鼠标肺中注入 4%甲醛或戊二醛/甲醛（1.5%/1.5%）混合物来固定组织块。根据体视学原理，从组织块中取样，用于石蜡、未经锇酸和醋酸铀酰处理的甲基丙烯酸乙二醇酯以及包括在脱水前用锇酸和醋酸铀酰处理的甲基丙烯酸乙二醇酯的包埋。通过对组织切面的尺寸变化进行体视学测量来研究收缩。结果表明，在石蜡包埋过程中，每个肺的切面面积收缩约 40%，在“简单”甲基丙烯酸乙二醇酯包埋过程中收缩约 30%，在锇酸/醋酸铀酰/甲基丙烯酸乙二醇酯包埋过程中收缩小于 3%。此外，还证明了含有戊二醛的固定剂在收缩方面的优势。总之，对于鼠标肺的体视学研究，建议使用含有戊二醛的固定剂，并在脱水前用锇酸和醋酸铀酰处理样品，然后用甲基丙烯酸乙二醇酯进行包埋。
