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

## TL;DR

Glyoxal is a better fixative than formaldehyde for preserving heart tissue structure and protein markers in mouse cardiac studies.

## Contribution

Glyoxal is shown to outperform formaldehyde in preserving antigenicity and structural integrity in murine cardiac tissues.

## Key findings

- Glyoxal fixation improves antibody penetration and antigen detection in thick cardiac tissues.
- Glyoxal enables better protein complex preservation for xIP-MS compared to formaldehyde.
- 3% glyoxal preserves heart structure, while 9% causes histological disruption.

## Abstract

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.

Unlabelled Image

•Glyoxal enhances IF signal, co-localization, and reduces staining artifacts.•3 % glyoxal preserves heart structure; 9 % disrupts histology and architecture.•Glyoxal improves antibody access to heart-resident cells via light crosslinking.•Light glyoxal crosslinking enables better protein extraction for cardiac xIP-MS.

Glyoxal enhances IF signal, co-localization, and reduces staining artifacts.

3 % glyoxal preserves heart structure; 9 % disrupts histology and architecture.

Glyoxal improves antibody access to heart-resident cells via light crosslinking.

Light glyoxal crosslinking enables better protein extraction for cardiac xIP-MS.