Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Department of Orthopedics, Trauma and Plastic Surgery, University of Leipzig, Leipzig, Germany.
Histol Histopathol. 2019 Aug;34(8):899-908. doi: 10.14670/HH-18-092. Epub 2019 Feb 13.
The histological processing of musculoskeletal tissue might be challenging. The alteration of tissue composition e.g. by calcification of soft tissue in the elderly, after trauma or surgical interventions makes the histological processing of fixed tissue difficult. Additional steps of decalcification are then needed that probably affect the staining quality. In the present work, the effects of different decalcification agents and the intermedium methyl benzoate on histological staining methods and immunohistochemistry have been compared. Acetabular labra were fixed with 4% paraformaldehyde, left untreated or decalcified using 30% ethylenediaminetetraacetic acid (EDTA; Chelaplex®) or 6% trichloroacetic acid (TCA) for 1-4 days to investigate the effects of decalcification duration. Moreover, samples were pretreated with methyl benzoate or conventionally paraffin embedded independent of decalcification procedure and duration. The specimens were evaluated using hemalaun-eosin, Azur II- methylene blue staining or immunohistochemistry against ankyrin B to visualize nerve fibers. Decalcification with Chelaplex® or TCA reduced cutting artifacts without affecting the tissue morphology and proteoglycan staining but decreased antigenicity in immunohistochemistry. Interestingly, methyl benzoate further reduced cutting artifacts without altering tissue morphology and elevated antigenicity for Chelaplex® decalcified tissue samples in immunohistochemistry. The decalcification with Chelaplex® or 6% TCA preserves tissue morphology and proteoglycan staining similar to non- decalcified tissue but facilitates section processing. In immunohistochemistry both decalcification agents decreased antigenicity. Chelaplex® decalcified, methyl benzoate treated samples yielded an improved antigenicity.
骨骼肌组织的组织学处理可能具有挑战性。组织成分的改变，例如老年人软组织的钙化、创伤或手术干预后，使得固定组织的组织学处理变得困难。然后需要进行额外的脱钙步骤，这可能会影响染色质量。在本工作中，比较了不同的脱钙剂和中间介质甲基苯甲酸对组织学染色方法和免疫组织化学的影响。髋臼唇用 4%多聚甲醛固定，未处理或用 30%乙二胺四乙酸（EDTA；Chelaplex®）或 6%三氯乙酸（TCA）脱钙 1-4 天，以研究脱钙时间的影响。此外，样品用甲基苯甲酸预处理或不依赖脱钙程序和时间常规石蜡包埋。使用苏木精-伊红、Azure II-亚甲蓝染色或针对锚蛋白 B 的免疫组织化学观察神经纤维来评估标本。Chelaplex®或 TCA 脱钙减少了切割伪影，而不影响组织形态和蛋白聚糖染色，但降低了免疫组织化学中的抗原性。有趣的是，甲基苯甲酸进一步减少了切割伪影，而不改变组织形态，并提高了免疫组织化学中 Chelaplex®脱钙组织样本的抗原性。Chelaplex®或 6%TCA 脱钙可保留组织形态和蛋白聚糖染色，类似于未脱钙组织，但可促进切片处理。在免疫组织化学中，两种脱钙剂均降低了抗原性。Chelaplex®脱钙、甲基苯甲酸处理的样品产生了改善的抗原性。
