Development of a H&E multi-agent-staining method for laboratory-microCT applied to endocrine glands of MENX rats

In histology, the widely used staining agents are hematoxylin and eosin (H&E), with hematoxylin marking cell nuclei and eosin staining the cytoplasm. By this, the color-coded information enables the early identification of histopathological changes using optical microscopy. However, the traditional histological process has significant drawbacks: the irreversible nature of tissue preparation often results in sample damage during dehydration, embedding, and sectioning, which can lead to the loss of crucial information. Moreover, standard microscopy techniques are limited to two-dimensional (2D) imaging, neglecting volumetric data crucial for detailed tissue analysis. X-ray imaging offers a non-destructive alternative, using contrast agents to enhance soft tissue visibility and allowing further investigations without compromising the sample. However, recently developed modified x-ray stains require adjustment for specific tissues, presenting a new challenge. Hematein can be chemically modified through the incorporation of high atomic number metals to enhance contrast in x-ray Imaging, whereas eosin staining can be augmented by increasing its concentration and acidifying the samples tissue. In this study, we aimed to demonstrate the feasibility of applying both modified hematoxylin and eosin stains to the same specimen sequentially, using a washing step with ethylenediaminetetraacetic acid (EDTA) to remove the hematein stain between scans. This novel approach preserves the distinct information provided by each stain, enabling comprehensive visualization in two separate micro-computed tomography (microCT) scans. The method was applied to biological samples from a rat strain spontaneously developing multiple endocrine tumors (MENX), provided by the Division of Neuroendocrinology at the Helmholtz Centre Munich. Specifically, the pituitary and adrenal glands of wild-type and MENX-affected rats were stained and imaged using the microCT system versaXRM-500 (ZEISS/xradia, Oberkochen Germany). The results revealed promising differentiation between healthy and affected tissues, with high-resolution imaging showing visible tumor formations, blood pools, and tissue degradation in diseased samples. This study highlights the potential of combining sequential H&E staining with microCT for enhanced tissue analysis and visualization of disease progression.