Delineation of the healthy rabbit heart by immunohistochemistry - A technical note

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining.