I am working on hepatoprotective activity. As a part of my study I have to conduct histopathological study of larvae. I went through different research papers. But each paper showing different procedure. Can any one share exact protocol for fixing zebrafish larvae by using 4 % PFA.
Aldehydes (formaldehyde, PFA) are standard histological fixatives. Immersion of the larvae in 4% PFA in 0.1M PBS (pH7.2, ~200 Osm, made fresh) overnight or longer at 4deg C should be sufficient. Use at least twice the volume of solution as your specimen(s). Make sure the larvae are euthanized just prior to fixation to reduce artifacts. Because the fish are very small you should not need any additional steps (e.g., flushing of fixative into buccal cavity, incisions in ventral body wall) to help with perfusion of the fixative.
I hope that helps,
Melissa
Here are some good sources for you:
- https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/fixation-strategies-formulations.html
- https://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives/
- 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 Part C: Toxicology & Pharmacology. http://dx.doi.org/10.1016/j.cbpc.2017.11.003
- Moore, J.L., Aros, M., Steudel, K.G., Cheng, K.C., 2002. Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis. Biotechniques 32, 296-298.
- Thavarajah, R., Mudimbaimannar, V., Elizabeth, J., Rao, U., Ranganathan, K., 2012. Chemical and physical basics of routine formaldehyde fixation. Journal of Oral and Maxillofacial Pathology 16, 400-405. http://dx.doi.org/10.4103/0973-029x.102496
- Tbakhi A, Totos G, Hauser-Kronberger C, et al. Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma. The American Journal of Pathology. 1998;152(1):35-41.
Hi there,
I fixed my zebrafish with 4% PFA solution with 1xPBS and 0.2% Tween-20 for performing in situ hybridisation with DIG-RNA labeled probes. After adding the 4% PFA, I kept the eppendorf tube containing the fish on its side overnight at 4C. Doing this will help keep the zebrafish from bending and give you really good samples to work with. Just remember to draw off the 4% PFA and don't let them over fixate.
Best of Luck
Thank you Melissa Chernick, Daniel Turkewitz, Emmanuel Ifeanyi Obeagu for your guidance,
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