Although relatively straightforward, the histological approach is strongly affected by the precise sequence and conditions in which the individual steps are carried out. As a result, the quality of histological images varies widely and is often poor in terms of contrast and border delineation. This introduces considerable subjectivity to AON/AAR measurements, compromising accuracy and, potentially, the validity of the conclusions.
This article describes an optimized protocol implemented in our laboratory, which reliably generates high-quality histological images. The key advantages include an entirely ex vivo staining method, greater color intensity in the remote myocardium (without smearing), and improved contrast and border delineation. Each step of the protocol is described in detail to assist other laboratories in implementing histology for in vivo I/R experiments.