Table 1.

Overview of different methods that can be applied in zebrafish models for ectopic mineralization.

Methods	Application	Stages of application in MO approach
MO injection	To evaluate the gene function by injecting synthetic anti-sense nucleotide oligomers	1–4 Cell stage embryos
Light microscopic observation	Phenotypic screening after injection (see Table 2).	Post-injection to morphant death
RNA rescue experiment	Validation of gene specificity by co-injection of MO and mRNA (encoding protein from the targeted locus of other species	1–4 Cell stages of embryos
Western blotting	Validation of the efficiency of TB MOs	After phenotypic confirmation, 1–4 dpf, until when effect of MO can be observed
PCR	Expression profiling of targeted gene	From 0- different time points
	Validation of the efficiency of SJ MOs	After phenotypic confirmation, 1–4 dpf, until when effect of MO can be observed
Quantitative real-time PCR	Expression profiling of targeted gene	From 0- different time points
	Validation of the efficiency of SJ MOs	After phenotypic confirmation, 1–4 dpf, until when effect of MO can be observed
Calcein staining	Fluorescent chromophores specifically bind to the calcified skeleton of live ZF embryos	5 dpf to morphant death
Alizarin red S	To identify calcium in tissue sections or whole mount embryos	4 dpf to morphant death
Alcian blue-Alizarin red double staining	Alcian blue stains cartilage blue and is used as a counterstaining to AR-S to distinguish cartilage and bone	4 dpf to morphant death
	Alizarin red stains as red in calcified matrix (calcified cartilage, bone)
IHC	To detect presence and localization of (mineralization-related) protein in tissue sections or whole mount embryos	0 hpf to morphant death
μCT imaging	Useful for skeletal analysis, used to understand developmental processes of three-dimensional embryos, embryo phenotyping, and quantitative modeling of development	5 dpf
ISH	To assess gene expression profiling in wild-type embryo and differential gene expression in morphant	0–4 dpf of embryos, as until 4 dpf effect of MO can be observed
MS	To analyze differential protein expression by measuring the mass-to-charge ratio	0–4 dpf, until MO effect can be observed
2D gel electrophoresis	To assess differential protein expression, where proteins are separated in the gel according to their isoelectric point	0–4 dpf, until MO effect can be observed
Microarray	Used to identify genome-wide expression of genes. In morphant differential expression of different gene can be identified	0–4 dpf, until MO effect can be observed
Transcriptome analysis	More sensitive compared to microarray, used to identify differential expression of transcripts. By this method closely homologous genes can be distinguished, alternatively spliced transcripts and non-coding RNAs can be characterized, and rare transcripts which are undetectable in microarray analysis can be detected	0–4 dpf, until MO effect can be observed
TUNEL staining	To assess in situ cell death in the whole mount embryo. TUNEL labels degraded DNA products enzymatically or by a fluorescent probe and stains apoptotic bodies	30 hpf–4 dpf, until MO effect can be observed
CMH2DCF staining	Used to determine oxidative stress or level of ROS in live embryos	0–4 dpf, until MO effect can be observed
MitoTracker Red CM-H2XRos	Used to determine mitochondrial membrane potentiality in live embryo	0–4 dpf, until MO effect can be observed
Chemical screening	Used to identify small chemicals which can rescue the morphant phenotype and can be predicted as a potential drug	0–4 dpf, until MO effect can be observed

MO, morpholino; PCR, polymer chain reaction; IHC, immunohistochemistry; ISH, in situ hybridization; MS, mass spectrophotometry; 2D, 2 dimensional.