Department of Tissue Engineering & Textile Implants, Applied Medical Engineering, University Hospital Aachen, Aachen, Germany.
Tissue Eng Part C Methods. 2012 Dec;18(12):976-83. doi: 10.1089/ten.TEC.2012.0159. Epub 2012 Jul 30.
In the immunohistochemical analysis of tissue-engineered structures, aggressive treatments for fixation and antigen retrieval can impair the quality of specimen staining and visualization.
We hypothesized that the adequate choice of fixative and antigen-retrieval method might improve the quality of immunohistochemical staining.
Tissue-engineered vascular grafts were fixed using formalin, Carnoy's, or HOPE(®) fixative. Antigen retrieval was performed where necessary and samples from each group were stained using hematoxylin and eosin to assess overall tissue preservation. For a set of proteins relevant to cardiovascular tissue development, immunohistochemical staining was applied to formalin-, Carnoy's-, and HOPE-fixed specimens to allow a comparative analysis.
In tissue-engineered constructs, antigen retrieval methods necessary after formalin fixation led to significant destruction of the overall tissue structure. Carnoy's fixation resulted in good overall tissue preservation and adequate results for immunohistochemical staining of alpha-smooth muscle actin (α-SMA), vimentin, type I collagen, elastin, and laminin. HOPE fixative led to a loosened tissue structure and a swollen appearance but showed adequate results for staining against type III collagen and elastin. Formalin fixation without antigen retrieval led to inadequate visualization of α-SMA, vimentin, type I- and type III collagen, and laminin.
Based on the present study, we recommend that Carnoy's fixative is employed for the preservation of tissue-engineered constructs to allow immunohistochemical analysis of type I- and type III collagen, elastin, laminin, α-SMA, and vimentin. However, it is clear that the technique requires optimization based on the particular tissue engineering application.
在组织工程结构的免疫组织化学分析中，固定和抗原修复的激进处理会损害标本染色和可视化的质量。
我们假设适当选择固定剂和抗原修复方法可能会提高免疫组织化学染色的质量。
使用福尔马林、卡诺氏或 HOPE（®）固定剂固定组织工程血管移植物。在必要时进行抗原修复，并用苏木精和伊红对每组样本进行染色，以评估整体组织保存情况。对于一组与心血管组织发育相关的蛋白质，对福尔马林、卡诺氏和 HOPE 固定的样本进行免疫组织化学染色，以进行比较分析。
在组织工程构建物中，福尔马林固定后需要进行抗原修复方法会导致整体组织结构的严重破坏。卡诺氏固定导致良好的整体组织保存，以及对α-平滑肌肌动蛋白（α-SMA）、波形蛋白、I 型胶原、弹性蛋白和层粘连蛋白的免疫组织化学染色有足够的结果。HOPE 固定剂导致组织结构松弛和肿胀，但对 III 型胶原和弹性蛋白的染色结果足够。未经抗原修复的福尔马林固定导致 α-SMA、波形蛋白、I 型和 III 型胶原以及层粘连蛋白的可视化不足。
基于本研究，我们建议使用卡诺氏固定剂保存组织工程构建物，以允许对 I 型和 III 型胶原、弹性蛋白、层粘连蛋白、α-SMA 和波形蛋白进行免疫组织化学分析。然而，很明显，该技术需要根据特定的组织工程应用进行优化。
