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

Liver diseases pose a big global health problem and liver failure may result from viral infection, overnutrition or tumors. Studying pathologic liver tissue demands for accurate and specific histological stainings and immunohistochemical labellings, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy liver stainings and labellings are required, to provide a baseline or reference for the pathological situation. Here, we used the liver tissue of a healthy rabbit and compared different histological key steps, such as paraffin embedding after formalin fixation versus cryopreservation; or an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or chromogenic with fluorescent detection system, respectively. Moreover, we provide images of serial sections, where we stained the same morphological structure with different markers, including collagen I, collagen III, fibronectin, α-SMA, elastin, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, ki67 for proliferating cells, and orcein (as negative control for pathological aberrations like Wilson disease). Differences between conditions were quantitatively assessed by measuring the colour intensity. Generally, we observed that cryosections exhibited a stronger signal intensity in immunohistochemically labelled sections than in paraffin sections; however, the strong staining got slurred, which sometimes hampered proper identification of morphological structures at higher magnifications. Moreover, there was a clear increase in signal intensity for paraffin sections when an AR step was performed compared to condition without AR. Results for mouse isotype staining as a negative control clearly supported those findings. Different stainings of the portal triad, the central vein and the bile ducts revealed a clear-cut distribution of extracellular matrix components, with prominent fibronectin and elastin around the lumen of the central vein as well as a patchy PAR-2 expression. As for the bile ducts, complete absence of α-SMA and PAR-2 was found at the margins, however, collagen I expression and elastin were positive and showed a strong signal. Like this, we provide useful and valuable reference images for researchers using the rabbit liver model. It may help to decide which of the immunohistochemical protocols are valuable to reach a certain aim and which protocols lead to the best visualization of the target structure