Teng Allen C T, Mehangrey Dev, Vandenbelt Ava, Vearncombe Karl, Callahan Justin D, Mistry Priya, Li Wenping, Reitz Cristine J, Hamed Omar, Roche Madison, Kuzmanov Uros, Fish Jason E, Epelman Slava, Gramolini Anthony O
Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Canada.
Department of Physiology, University of Toronto, Canada.
J Mol Cell Cardiol Plus. 2025 May 11;12:100454. doi: 10.1016/j.jmccpl.2025.100454. eCollection 2025 Jun.
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.
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.
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.
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.
免疫荧光（IF）是评估组织标本中组织学和生化变化的一项重要技术。IF的一个关键步骤是样本固定，通常使用基于甲醛的固定剂来实现，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，这可能会改变抗原性并产生假象。本研究调查了乙二醛（一种二碳二醛）作为用于小鼠心脏组织IF和交联免疫沉淀-质谱分析（xIP-MS）应用的潜在替代固定剂。
将不同浓度和固定时间的乙二醛与4% PFA进行比较。使用苏木精和伊红（H&E）染色评估组织结构完整性，同时通过荧光显微镜评估心肌细胞中的抗原保存情况。还研究了心脏驻留细胞（包括心脏成纤维细胞、平滑肌细胞和内皮细胞）的免疫荧光。在乙二醛固定的小鼠心脏中通过磷酸受磷蛋白（PLN）免疫沉淀进行xIP-MS分析，随后进行质谱分析。
乙二醛在心脏组织结构和肌纤维完整性保存方面与PFA相当，但在抗原保留和蛋白质检测方面表现更优。对肌浆网标志物（SERCA2和PLN）、闰盘蛋白（N-钙黏蛋白和Cx43）以及收缩蛋白（F-肌动蛋白和肌球蛋白重链）进行了荧光成像。定量图像分析证实，乙二醛增强了抗体在较厚组织（30μm）中的渗透，并维持了各种心脏驻留细胞标志物的抗原性。乙二醛固定通过使PLN与其相关蛋白复合物轻度交联从而实现xIP-MS，能够鉴定小鼠心脏中新型的PLN相互作用蛋白。
我们的研究结果强调了乙二醛在心脏生物化学研究中作为PFA的优质替代品的实用性，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存方面有所改进。
