Glyoxal is a superior fixative to formaldehyde in promoting antigenicity and structural integrity in murine cardiac tissues

Allen C.T. Teng , Dev Mehangrey , Ava Vandenbelt , Karl Vearncombe , Justin D. Callahan , Priya Mistry , Wenping Li , Cristine J. Reitz , Omar Hamed , Madison Roche , Uros Kuzmanov , Jason E. Fish , Slava Epelman , Anthony O. Gramolini
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Abstract

Background

Immunofluorescence (IF) is an essential technique for evaluating histological and biochemical changes in tissue specimens. A critical step in IF is sample fixation, typically achieved using formaldehyde-based fixatives, such as 4 % paraformaldehyde (PFA) or 10 % formalin. However, these fixatives are prone to over-fixation, which can alter antigenicity and promote artifacts. This study investigated glyoxal, a two‑carbon dialdehyde, as a potential alternative fixative for murine cardiac tissues for IF and crosslinking immunoprecipitation-mass spectrometry (xIP-MS) applications.

Methods

Various concentrations and fixation durations of glyoxal were compared with 4 % PFA. Tissue structural integrity was assessed using Hematoxylin and Eosin (H&E) staining, while antigen preservation in cardiomyocytes was evaluated through fluorescent microscopy. Immunofluorescence of cardiac resident cells, including cardiac fibroblasts, smooth muscle cells, and endothelial cells were also investigated. xIP-MS assays were carried by phospholamban (PLN) immunoprecipitation in glyoxal-fixed mouse hearts, followed by mass spectrometry analysis.

Results

Glyoxal showed comparable preservation of cardiac tissue architecture and myofiber integrity to PFA, but with superior antigen retention and protein detection. Fluorescent imaging was performed for sarcoplasmic reticulum markers (SERCA2 and PLN), intercalated disc proteins (N-Cadherin and Cx43), and contractile proteins (F-Actin and MyHC). Quantitative image analysis confirmed that glyoxal enhanced antibody penetration in thicker tissues (30 μm) and maintained the antigenicity of various cardiac resident cell markers. Glyoxal fixation allowed for xIP-MS by lightly crosslinking PLN with its associated protein complexes, enabling the identification of novel PLN-interacting proteins in mouse hearts.

Conclusion

Our findings underscore the utility of glyoxal as a superior alternative to PFA in cardiac biochemistry research, offering improvements in the preservation of tissue morphology, antigen detection, and protein complex conservation in murine cardiac tissues.
乙二醛在促进小鼠心脏组织抗原性和结构完整性方面是一种优于甲醛的固定剂
免疫荧光(IF)是评估组织标本组织学和生化变化的重要技术。IF的关键步骤是样品固定,通常使用基于甲醛的固定剂,如4%多聚甲醛(PFA)或10%福尔马林。然而,这些固定剂容易过度固定,这可能改变抗原性和促进伪影。本研究研究了乙二醛,一种二碳双醛,作为小鼠心脏组织的潜在替代固定剂,用于IF和交联免疫沉淀-质谱(xIP-MS)应用。方法采用4% PFA对乙二醛不同浓度和固定时间进行比较。采用苏木精和伊红(H&;E)染色评估组织结构完整性,通过荧光显微镜评估心肌细胞中的抗原保存情况。心脏常驻细胞,包括心脏成纤维细胞、平滑肌细胞和内皮细胞的免疫荧光也进行了研究。采用磷蛋白(PLN)免疫沉淀法在乙二醛固定小鼠心脏中进行iip - ms检测,然后进行质谱分析。结果乙二醛对心肌组织结构和肌纤维完整性的保护与PFA相当,但具有更强的抗原保留和蛋白质检测能力。对肌浆网标记物(SERCA2和PLN)、嵌层盘蛋白(N-Cadherin和Cx43)和收缩蛋白(F-Actin和MyHC)进行荧光成像。定量图像分析证实,乙二醛增强了抗体在较厚组织(30 μm)中的渗透,并保持了各种心脏驻留细胞标记物的抗原性。乙二醛固定允许通过轻度交联PLN与其相关蛋白复合物进行xIP-MS,从而鉴定小鼠心脏中新的PLN相互作用蛋白。结论我们的研究结果强调了乙二醛作为PFA的优良替代品在心脏生物化学研究中的应用,在小鼠心脏组织的组织形态保存、抗原检测和蛋白质复合物保护方面提供了改进。
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来源期刊
Journal of molecular and cellular cardiology plus
Journal of molecular and cellular cardiology plus Cardiology and Cardiovascular Medicine
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