CardiLect: A combined cross‐species lectin histochemistry protocol for the automated analysis of cardiac remodelling

Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability. Direct comparisons between the two methods are lacking from the literature.In this study, we compared antibody- and lectin-based methods for the histological assessment of cardiomyocyte CSA (with the use of anti-laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti-CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy-to-use combined lectin histochemistry protocol (WGA and ILB4, 'CardiLect' protocol) for the histological assessment of cardiac remodelling. The lectin-based approach has been evaluated on a cross-species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script ('CardiLect Analyser') for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P = 0.0098, rS = 0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P = 0.0402, rS = -0.6364). Microvascular density showed significant negative correlation with LV mass (P = 0.0055, rS = -0.7622) and significant positive correlation with EF (P = 0.0106, rS = 0.7203).The described combined lectin histochemistry protocol with the provided ImageJ script is an easy-to-use, cost-effective, cross-species approach for the histological assessment of cardiac remodelling.