Zupančič Daša, Terčelj Marjeta, Štrus Bojan, Veranič Peter
Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia.
Department of Pulmonary and Allergic Diseases, University Medical Centre Ljubljana, Zaloška cesta 2, 1000, Ljubljana, Slovenia.
Protoplasma. 2017 Sep;254(5):1931-1939. doi: 10.1007/s00709-017-1085-0. Epub 2017 Feb 10.
Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with "sucrose impregnation prior to freezing" method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.
大多数人类和动物活检样本通常被包埋在石蜡中，因为这能使病理学家或研究人员获得出色的形态结构，并且便于储存。然而，在许多情况下，石蜡切片中无法检测到感兴趣的抗原。对于良好的免疫组织化学而言，另一种选择是制备冰冻切片，冰冻切片通常能较好地保存抗原，并且常用于免疫荧光检测。然而，冰冻切片往往无法为病理评估提供有效的组织和细胞形态细节。为了在冷冻后获得良好的抗原保存效果并改善组织和细胞形态，我们测试了三种不同的固定和冷冻方法，并将它们与常规甲醛固定和石蜡包埋进行比较。作为模型系统，我们选择了大鼠膀胱和气管的上皮组织。对所有样本进行苏木精和伊红染色以及针对紧密连接蛋白ZO-1和中间丝细胞角蛋白7的抗体进行免疫荧光检测。采用“冷冻前蔗糖浸渍”方法在形态和免疫荧光之间取得了最佳平衡。此外，与标准石蜡切片制备相比，该方法速度更快。为了检验我们研究的临床相关性，该方法被用于肿瘤性尿路上皮和支气管黏膜病变的人类活检样本。除了获得良好的免疫荧光结果外，这些样本的形态也得到了很好的保存。因此，我们建议在其他临床和兽医应用中应进一步采用冷冻前蔗糖浸渍的冰冻切片制备方法，因为它能实现良好的形态和抗原保存。
