lines and maintenance
Zebrafish (Danio rerio) were bred, raised and
maintained at 28.5 C under standard conditions as described (ref). Embryo
staging was done by using both timing (hours post fertilization, hpf) and
morphological features as previously described (ref). For in situ hybridization
analysis of embryos older than 24hpf, embryos were raised in 1-phenyl-2-
thiourea (PTU) (0.003% in Embryo medium), to inhibit pigment formation (ref).
Zebrafish handling was in strict accordance with good animal practice as
defined and all experiments were performed using protocols approved by the
Institutional Animal Ethics Committee (IAEC) of the CSIR-Institute of Genomics
and Integrative Biology, India. The zebrafish lines used in this study are
Tuebingen (TU), used as the wild-type strain and tfr2f6 mutant strain.
A small portion of fin
tissue was clipped from the adult zebrafish tail and collected into PCR lysis
buffer containing proteinase K for isolating genomic DNA. Two sets of allele
specific primer (Supplementary table X)
of tfr2 were used to determine the genotype of the fish.
The RNA was isolated from embryos (TU) and tfr2f6
mutant embryos and cDNA was prepared and two sets of allele specific primer (Supplementary table X) of tfr2 were used to
determine the genotype of the fish.
Whole mount Prussian blue staining for iron
24 hpf and 7 dpf embryos
were fixed in 4% paraformaldehyde (PFA) for overnight. The embryos were rinsed
with phosphate buffered saline with tween 20 (PTW) and followed by dehydration
with various percentage methanol and kept at -20 for overnight fixation.
Rehydrate the embryos with 50% methanol and PTW and followed by PTW wash. Then
embryos were incubated with iron staining solution (HT-20 Sigma Aldrich) for 30 mins at room temperature (RT).
The embryos were rinsed 3 times in PTW, followed by incubation in 0.3%
H2O2 in methanol for 20 minutes at RT. The embryos were rinsed twice in PTW and
then incubated in DAB substrate (Sigma Fast
3,3_-DAB tetrahydrochloride with cobalt chloride enhancer (Cat no. D-0426,
Sigma-Aldrich) for 5 mins at room temperature. Photograph of embryos
were captured using Zeiss (Stemi 2000CVR) bright field microscope at 5X
magnification (with AxiocamICc1
Chemical screening of small molecule modulators of hepcidin
A library compound was
used for hepcidin screening assay. A pool of 10 embryos were taken in 48 well
plate with 1 ml of E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM
MgSO4) in each well. The concentration of 10uM
of the compound were added at 56 hpf of WT (TU) embryos and incubated at 28
degree for 16 hours. At 72 hpf phenotype of the treated embryos were checked by
bright field microscopy and embryos were fixed in 4% paraformaldehyde (PFA) and
further processed for whole mount RNA in situ hybridization of hepcidin.
Whole mount RNA in situ hybridization
Whole mount RNA in situ hybridizations were performed in 3dpf
embryos as described. (references). Hepcidin and fabp10a ribo
probe were used in this study were kind gift by Paula G. Fraenkel. Photograph of embryos were captured using Zeiss (Stemi 2000CVR) bright
field microscope at 5X magnification (with AxiocamICc1).
Realtime quantitative PCR
Zebrafish embryos (N=
15-20) and tissue were fixed in RNAiso Plus (Takara, cat no:xxxx) and total RNA was isolated by
previously described method. (Ref) Purity
was assessed by 260/280 nm absorbance
ratios in nanodrop (instrument model) and
quality was monitored by agarose gel electrophoresis. On average 1ug RNA was used for cDNA synthesis. cDNAs were subjected to
PCR amplification with serial dilutions to check the amplification efficiency
of primers used in qRT PCR. c DNA was synthesized by using QuantiTect
Reverse Transcription kit (Qiagen
cat no ) and Quantitative real time PCR was carried
out as described (ref)
on The LightCycler® 480 System. For quantification, the relative
standard curve method was used (as described by the manufacturer) to generate
raw values representing arbitrary units of RNA transcript. Each experiment was
performed on three independent occasions in biological triplicates of pooled
embryos (in every experiment). Statistical analyses on normalized data were
performed using 2-??CT algorithm (known as the delta-delta-Ct or
ddCt algorithm) (ref).
All genes were normalized against RPL13a unless mentioned otherwise. The Graph
pad prism software was used for the analysis. For statistical significance of
the data, p-values were calculated by performing unpaired student t-test. All
primers used in this study are listed in Supplementary Materials Tables xxx
Zebrafish liver tissue
were collected and homogenized in NP40 lysis buffer (invitrogen). Protein was estimated by using BCA protein assay
method by using Pierce™ BCA Protein Assay Kit (Cat
No :23225, Life technology) . Then immunoblot was performed as described
(ref ). All samples (50 ug of
protein per lane) were analyzed by SDS-PAGE on 10% gels by standard procedures.
Following transfer of the proteins onto PVDF membrane (Milipore), the blots were saturated with 5% BSA in Tris-buffered
saline (PBS) containing 0.1% (v/v) Tween-20 (TBST) for 2 hour, and probed
overnight with a 1:1000 diluted primary antibodies (Anaspec). After three washes with TBST, the
blots were incubated with 1:10000 diluted HRP conjugate anti-rabbit IgG (Cell
Signalling) for 1.5 hour. The peroxidase signal was detected by EMD Millipore Immobilon™ Western Chemiluminescent HRP
Small molecule injection
in adult zebrafish
Adult animal were anesthetized with tricane and SC-514 and PDTC
(23 ?g g?1) were injected into abdominal cavity with vehicle DMSO and control
animal were injected with DMSO. Animals were revived into the water. After 2.5
hours of injection, animal were again anesthetized in tricane, and liver were
dissected out and collected into 100 ?l of RNAiso Plus (Takara, cat no:xxxx) mechanically homogenized
and total RNA was isolated as previously described.