PUBLICATION
Evaluating the Effects of Various Decalcification Protocols on Immunohistochemical Staining in Zebrafish (Danio rerio)
- Authors
- Meritet, D.M., Spagnoli, S.T., Fischer, K.A., Löhr, C.V.
- ID
- ZDB-PUB-190425-16
- Date
- 2019
- Source
- Zebrafish 16(3): 280-290 (Journal)
- Registered Authors
- Löhr, christiane
- Keywords
- CalExII, Dietrich's, decalcification, immunohistochemistry, neutral buffered formalin, zebrafish
- MeSH Terms
-
- Animals
- Decalcification Technique/methods*
- Staining and Labeling/methods*
- Tissue Fixation*/methods
- Zebrafish*
- PubMed
- 31017539 Full text @ Zebrafish
Citation
Meritet, D.M., Spagnoli, S.T., Fischer, K.A., Löhr, C.V. (2019) Evaluating the Effects of Various Decalcification Protocols on Immunohistochemical Staining in Zebrafish (Danio rerio). Zebrafish. 16(3):280-290.
Abstract
Fixation and decalcification can alter protein structure in tissues, influencing the efficacy of primary antibodies routinely used in immunohistochemical (IHC) staining. Histologic examination of zebrafish requires both processes, making staining and analysis potentially challenging. Here, we investigated the effects of common fixation and decalcification protocols on IHC staining in zebrafish. We also identified zebrafish-reactive and -specific antibodies for use in research and diagnostics. For several of the antibodies, time spent in Dietrich's fixative containing 2% glacial acetic acid or 3.4% formaldehyde followed by decalcification with ethylenediaminetetraacetic acid (EDTA) significantly impacted IHC staining quality, particularly regarding staining intensity. Protocols utilizing shorter fixation times produced higher-quality stains. In addition, individual markers were variably affected by the type of fixative. Dietrich's fixative significantly reduced staining quality for the "neural" markers: glial fibrillar acidic protein, chromogranin A, S100. A negative time-dependent effect of fixation on staining quality was found for several antibodies: muscle actin (Dietrich's only), cytokeratin AE1/AE3, chromogranin, and S100. Neither decalcification protocol had a statistically significant negative time-dependent effect on staining quality. Based on our results, we suggest shorter fixation and decalcification protocols to best preserve IHC staining quality as well as recommend deliberate selection of the fixative used depending on the protein of interest.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping