For chronic developmental exposures
(Figures 2 , 3 , and 4 B), zebrafish embryos
were obtained from natural group spawning and age-matched within the
first hour of fertilization. Enzymatic dechorionation took place at
4 h postfertilization (hpf), where ∼800 embryos were placed
in a round glass plate (10 cm diameter) with 25 mL system water (same
water in which fish were raised) to which 50 μL of 63.6 mg/mL
(∼11.12 U) Pronase E (protease from Streptomyces griseus, ≥ 3.5 U/mg, P5147 Sigma-Aldrich, St. Louis, Missouri) was
added. Constant manual plate agitation took place for 6 min following
a wash with 2 L of system water.49 (link) Dechorionated
embryos were randomly placed in individual wells in 96-well plates
(Falcon, Fisher Scientific, Hampton, New Hampshire) with 100 μL
embryo medium (EM).50 Chemical stocks (1000×)
were diluted to 2× in EM, and 100 μL was added to each
well, resulting in final concentrations 0.1, 0.3, 1, 3, and 10 μM
in 0.1% DMSO. Each experimental group included 16 fish per spawning
from three separate spawning events, resulting in a total of n = 48 per group. Plates were covered in Parafilm M (Bemis,
North America, Neenah, Wisconsin) and placed in a 28.5 °C light-controlled
(14 h light (∼300 lx):10 h dark) incubator for static waterborne
exposure through 5 dpf.
For acute developmental exposures (Figure 4 A), embryos were
also obtained by natural group spawning. Embryos were raised in Petri
dishes without chemically induced dechorionation until 4 dpf, when
they were transferred to 96-well plates with 100 μL EM. At 5
dpf, exposures were conducted identically to chronic developmental
exposures (100 μL of compounds at 2× of the final concentration
was added to each well) with the exception that behavioral assessments
were conducted immediately after addition of chemical compounds to
wells containing zebrafish larvae.
(
were obtained from natural group spawning and age-matched within the
first hour of fertilization. Enzymatic dechorionation took place at
4 h postfertilization (hpf), where ∼800 embryos were placed
in a round glass plate (10 cm diameter) with 25 mL system water (same
water in which fish were raised) to which 50 μL of 63.6 mg/mL
(∼11.12 U) Pronase E (protease from Streptomyces griseus, ≥ 3.5 U/mg, P5147 Sigma-Aldrich, St. Louis, Missouri) was
added. Constant manual plate agitation took place for 6 min following
a wash with 2 L of system water.49 (link) Dechorionated
embryos were randomly placed in individual wells in 96-well plates
(Falcon, Fisher Scientific, Hampton, New Hampshire) with 100 μL
embryo medium (EM).50 Chemical stocks (1000×)
were diluted to 2× in EM, and 100 μL was added to each
well, resulting in final concentrations 0.1, 0.3, 1, 3, and 10 μM
in 0.1% DMSO. Each experimental group included 16 fish per spawning
from three separate spawning events, resulting in a total of n = 48 per group. Plates were covered in Parafilm M (Bemis,
North America, Neenah, Wisconsin) and placed in a 28.5 °C light-controlled
(14 h light (∼300 lx):10 h dark) incubator for static waterborne
exposure through 5 dpf.
For acute developmental exposures (
also obtained by natural group spawning. Embryos were raised in Petri
dishes without chemically induced dechorionation until 4 dpf, when
they were transferred to 96-well plates with 100 μL EM. At 5
dpf, exposures were conducted identically to chronic developmental
exposures (100 μL of compounds at 2× of the final concentration
was added to each well) with the exception that behavioral assessments
were conducted immediately after addition of chemical compounds to
wells containing zebrafish larvae.
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