Tissue Engineering: Selecting the Optimal Fixative for Immunohistochemistry Sabine Koch, 1, * Nadine Stappenbeck, B.Sc., 1, * Christian G. Cornelissen, M.D., 1,2 Thomas Cormac Flanagan, Ph.D., 3 Petra Mela, Ph.D., 1 Jo ¨ rg Sachweh, M.D., 1 Benita Hermanns-Sachweh, M.D., 4 and Stefan Jockenhoevel, M.D. 1 Background: In the immunohistochemical analysis of tissue-engineered structures, aggressive treatments for fixation and antigen retrieval can impair the quality of specimen staining and visualization. Hypothesis: We hypothesized that the adequate choice of fixative and antigen-retrieval method might improve the quality of immunohistochemical staining. Methods: Tissue-engineered vascular grafts were fixed using formalin, Carnoy’s, or HOPE Ò 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. Results: In tissue-engineered constructs, antigen retrieval methods necessary after formalin fixation led to sig- nificant 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 (a-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 a-SMA, vimentin, type I- and type III collagen, and laminin. Conclusion: 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, a-SMA, and vimentin. However, it is clear that the technique requires optimization based on the particular tissue engineering application. Introduction H istology and immunohistochemistry are important tools to assess microscopic structure, cell organization, and function of tissues, either native or tissue-engineered. Since tissue is subject to autolysis, specific fixation methods have been developed to stabilize the intracellular and ex- tracellular components. 1 The appropriate fixation method should preserve cells as they naturally occur, retaining all morphological details and preventing antigen destruction as much as possible. Applying a fixation method always im- plicates a compromise between tissue preservation and de- struction. A modified macroscopical structure, inactivated enzymes, 2 conformational changes in the protein structure, 3 new antigen profiles 4 , and a tissue with altered biochemical composition are all potential outcomes of fixation. 5 Formalin is the most widely used fixative for histological and immunohistochemical analysis of tissues. 4,5 Formalin fixation is well established in routine clinical applications and its excellent results in most standard applications render it the gold standard. Formalin is a cross-linking fixative and chemically binds to protein molecules after denaturing them. 1 This aggressive procedure masks the antigen epitopes of pro- teins, thus rendering them inaccessible for further immuno- histochemical marking. Thus, antigen retrieval by enzymatic pretreatment 6,7 or heat conditioning 8,9 is mandatory 10 for im- munohistochemical analysis of formalin-fixed tissues, further adding to the aggressive nature of the method. 11,12 Due to the delicate nature of tissue-engineered constructs prepared in vitro, they are prone to damage by this well- established standard fixation procedure. For this reason, the analysis of tissue-engineered constructs requires more gentle 1 Department of Tissue Engineering & Textile Implants, Applied Medical Engineering, University Hospital Aachen, Aachen, Germany. 2 Medizinische Klinik I and Pneumology, University Hospital Aachen, Aachen, Germany. 3 School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, Dublin, Ireland. 4 Department of Pathology, University Hospital Aachen, Aachen, Germany. *These two authors equally contributed to this work. TISSUE ENGINEERING: Part C Volume 18, Number 12, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2012.0159 976