PUBLICATION
Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish
- Authors
- Copper, J.E., Budgeon, L.R., Foutz, C.A., van Rossum, D.B., Vanselow, D.J., Hubley, M.J., Clark, D.P., Mandrell, D.T., Cheng, K.C.
- ID
- ZDB-PUB-171122-7
- Date
- 2017
- Source
- Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 208: 38-46 (Journal)
- Registered Authors
- Cheng, Keith C.
- Keywords
- Atlas, Decalcification, Embedding, Fixation, Histology, Sectioning, Staining, Virtual slide
- MeSH Terms
-
- Animals
- Calcium Chelating Agents/chemistry
- Decalcification Technique*
- Edetic Acid/chemistry
- Fixatives/chemistry
- Formaldehyde/chemistry
- Hydrogen-Ion Concentration
- Image Processing, Computer-Assisted
- Microscopy
- Microtomy
- Paraffin Embedding/methods*
- Specimen Handling/methods*
- Staining and Labeling
- Tissue Fixation/methods*
- Zebrafish/embryology*
- PubMed
- 29157956 Full text @ Comp. Biochem. Physiol. C Toxicol. Pharmacol.
Citation
Copper, J.E., Budgeon, L.R., Foutz, C.A., van Rossum, D.B., Vanselow, D.J., Hubley, M.J., Clark, D.P., Mandrell, D.T., Cheng, K.C. (2017) Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP. 208:38-46.
Abstract
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 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, 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping