Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish.

In recognition of the importance of zebrafish as a model organism for studying human disease, we have created zebrafish content for a web-based reference atlas of microanatomy for comparing histology and histopathology between model systems and with humans (http://bio-atlas.psu.edu). Fixation, decalcification, embedding, and sectioning of zebrafish were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21°C for 24h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35M produced effective decalcification in 21-day-old juveniles through adults (≥~3Months). To improve section plane consistency in sets of larvae, we have developed new array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue discontinuity in sections, a common barrier to creating quality sections of zebrafish, was minimized by processing and embedding the formalin-fixed zebrafish tissues in plasticized forms of paraffin wax, and by periodic hydration of the block surface in ice water between sets of sections. Optimal H&E (Hematoxylin and Eosin) staining was achieved through refinement of standard protocols. High quality slide scans produced from glass histology slides were digitally processed to maximize image quality, and experimental replicates posted as full slides as part of this publication. Modifications to tissue processing are still needed to eliminate the need for block surface hydration. The further addition of slide collections from other model systems and 3D tools for visualizing tissue architecture would greatly increase the utility of the digital atlas.

High-throughput zebrafish histology.

The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism's small larvae also makes it a potentially important model for toxicological analysis. Studying histological phenotypes is greatly enhanced by high-throughput methods of histology. Here, we describe details of high-throughput histology of the zebrafish using larval arrays, along with recent advances in mold design and discussion of work in progress that will lead to easier ways for people in the field to more rapidly score phenotypes in arrays. These detailed descriptions, together with the troubleshooting guide, should enable any laboratory with ties to a histology facility to perform high-throughput histology of zebrafish.