TABLE 3.

Troubleshooting table

Step	Problem	Possible reason	Solution
8	Fish larvae are not reliably detected by the Tecan microplate reader (well appears empty)	Convex/concave shape of the liquid:air interface refracts both excitation and emission light, decreasing acquired signal	Check the total volume of wells and if necessary adjust to ~330 μl to provide a consistent flat liquid surface before the scan. Make sure that U-shaped 96-well plates were used
		Irregular placement of zebrafish within wells—i.e., organ or tissue of interest is irregularly placed and potentially missed in regional scans	Consider using the Tecan orbital shaking function before the scan for a more uniform larva orientation in each well. Alternatively, determine which type of 96-well plate (U-bottom, V-bottom, flat-bottom) permits the most uniform placement of your larvae (age- and tissue-specific)
		Fish larvae are not fully anesthetized	Ensure that the final concentration of clove oil is accurate (0.02% final concentration of clove oil). Wait for 15 min before the scan; verify the lack of a startle response by tap-ping the edge of the plate, and check for movement of larvae
		‘Z-Position’ setting (μm) of the 96-well-plate scan is not optimal	Complete a ‘Z-Position’ scan to re-establish/verify optimal settings. Take the average of the depths from each well that provided maximal signal; this is your new ‘Manual Z-Position’
		Inappropriate parameter settings	Settings may need to be adjusted for maximal signal detection
		Suboptimal Tecan Excitation, Emission and/or Bandwidth settings are used	Perform Excitation and Emission ‘Fluorescence Intensity Scans’ (±10 nM) centered at published Ex/Em maxima for your fluoro-phore (±10 nm) to maximize Signal:Background ratio
		The fluorescent protein is not expressed at high-enough levels	Use an alternative transgenic line or promoter that drives higher transgene expression. Consider CRIPSR knock-in versus Tol2-mediated transgenesis, or a bi-partite system to enhance expression levels
		PTU (photo-labile) is degraded and embryos have become pigmented	Protect PTU stock solution from light and transfer the eggs to PTU/E3 medium before 16 hpf. Ensure that 96-well plates are uniformly exposed to light—i.e., avoid stacking plates
25	Insufficient egg quantity (low fecundity)	Aquaculture system conditions are not favorable for fish health	Check the levels of the following: pH, nitrate, nitrite, ammonia, salinity, water temperature, and daily exchange rate of system water; verify consistent light:dark cycle. Check with your aquaculture system manufacturer for recommended settings
		Fish are aged or their age is not optimal for mating	Maintain replacement breeder stocks to rotate new fish into the screen every 6 months (3-to 6-month-old adults provide highest fecundity)
29	Insufficient egg quality	Unfertilized eggs (opaque/white)	Increase the male:female ratio or consider replacing aged males (9+ months) with younger (3+ months) males
		Robust mycotic (fungal) growth in plates	Thoroughly rinse the eggs during collection and remove detritus. Use embryo medium that includes methylene blue (anti-mycotic) for newly fertilized eggs during the first 16–24 h
49	Splashing of drug compound (of varying concentrations) between wells when the fish droplet is dispensed from COPAS	Droplet size is too large	Adjust COPAS ‘width’ metric (in milliseconds) to obtain an approximately 50-μl droplet size. Dispense larvae into a well that is 1/3 full. For details, see Supplementary Figure 5
		Droplet placement is not centered in the well	Recalibrate COPAS plate coordinates to recenter the droplets. In addition, verify that COPAS ‘Sorter’ pressure (psi) is optimized for your other component pressures (‘Sheath’, ‘Sample’, ‘Clean’)
	No fish in the wells	Component pressures (‘Sheath’, ‘Sample’, ‘Clean’, ‘Sorter’) and/or gating/sorting parameters (Supplementary Fig. 5) are not optimized	Pass several hundred nontransgenic, and then transgenic, larvae through the COPAS to separate fish from nonfish (gating) and transgenic from nontransgenic (sorting). Adjust component pressures and/or gating/sorting windows, and repeat this process until optimal
	Multiple fish in the wells	Fish concentration in ‘Top Cup’ of COPAS (where large quantities of fresh larvae are placed) is too high	Decrease the concentration of fish in ‘Top Cup’; 1–2 fish per milliliter is ideal
	Embryo dispension rate is slow	Not enough sample pressure in COPAS	Increase the ‘Top Cup’ and/or ‘Sample Cup’ pressure in COPAS
		Fish concentration in ‘Top Cup’ of COPAS (where large quantities of fresh larvae are placed) is too low	Increase the concentration of fish in ‘Top Cup’, 1–2 fish per milliliter is ideal
51	Evaporation proximal to the edges of the plate	There is not enough humidity where the plates are stored. This will lead to an ‘edge-effect’ in which signal acquired from the edges of the plate is decreased as compared with that from more central wells	Check the humidity level and consider storing plates in a humidified incubator
	Condensation on lids of plates, leading to mixed concentrations of serially titrated drug compounds	Frequent temperature fluctuations (e.g., incubator door opened frequently)	Consider storing the plates in a humidified incubator, and make sure that the incubator door seals well
62	R GUI—‘ARQiv package graphical user interface (GUI)’ is not functional	‘well.label.csv’ file is incorrectly formatted	This file identifies the location (96-well plate format) of positive/negative controls, as well as that of drug compounds. Verify the accuracy of this template file
		Software needs to be updated and/or the package is missing	Ensure that R and R Studio are up to date. Re-download the ARQiv package from Github
63	Dead embryos after treatment	Drug concentration is too high or drug is toxic to zebrafish	Adjust the drug serial dilution concentration(s) to potentially retest this compound
		Timing of chemical exposure is wrong	Establish appropriate exposure times with a positive control drug (i.e., length of exposure, or specific to developmental window)