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 a web-based reference atlas of zebrafish microanatomy for comparing histology and histopathology between model systems and with humans ( http://bio-atlas.psu.edu ). Fixation, decalcification, embedding, and sectioning were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21 °C for 24 h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35 M produced effective decalcification in 21-day-old juveniles through adults (≥~3 Months). To improve section plane consistency in sets of larvae, our lab developed agarose array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue section discontinuity, a common barrier to creating quality sections of zebrafish but not mammalian tissues, 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 make web-based images similar to what is seen under the highest quality microscopes. Future directions for our bio-atlas include further improvements in tissue processing and the addition of slide collections from other model systems as well as 3D tools for visualizing tissue architecture.