In this video image-based high content screening is performed to assess small molecule based modulation of the fibroblast growth factor Rasm map kinase pathway In zebrafish embryos transgenic male and wild type female zebrafish are mated in a breeding cage. Fertilized eggs fall at the bottom and are collected and transferred to a Petri dish. The embryos are incubated overnight, then placed into individual wells of a 96 well round bottom plate.
The small molecule BCI is added to hyper activate FGF signaling and the plate is incubated for six hours. Images from each will are captured with a confocal plate reader and analyzed using a self-designed algorithm which reports fluorescent intensities in specific embryonic structures. Hi, I'm Hiba Kado from the laboratory of Dr.Michael Chang at the Department of Microbiology and Molecular Genetics at the University of Pittsburgh.
Hi, I'm Andrea Spock from the University of Pittsburgh Drug Discovery Institute. Today we will show you the procedure for breeding, selecting and arraying zebra fish embryos for chemical treatment. In a 96 well plate, we will use transgenic embryos that report on fiberglass growth factor activities and treated with a molecule that hyper activate this past white.
We will show you how we use high content screening methodology to automatically acquire, analyze, and quantify fibroblast growth factor activity in transgenic zebra fish embryos. This procedure will highlight platform technology to utilize transgenic zebra fish and high throughput chemical screens. So let's get started.
Two days prior to treatment, identify homozygous male transgenic zebrafish and place them into individual mating tanks. Add separators to the tanks, then add a wild type female fish to each tank and leave the fish in the mating cages overnight at 28 degrees Celsius. The next morning, remove the separator After one hour, move the fish to a new tank and pour the embryos into a plastic sieve.
Transfer the embryos to 100 millimeter dishes with E three solution and incubate at 28 degrees Celsius for six to eight hours. Following the incubation, examine the embryos under a stereo microscope. Embryos suitable for experiments will be undergoing gas.
At this time, remove any unfertilized embryos, which will remain at the single cell stage. Also, remove non-viable embryos which appear opaque or malformed. Then add fresh E three solution and incubate overnight at 28 degrees Celsius.
The next day, place the embryos under a fluorescent stereo microscope to sort the transgenic embryos for similar stage and uniform. GFP expression 24 HPF embryos show a visible mid hind brainin boundary with intense GFP expression and well-formed eyes with GFP positive dorsal retinas. These embryos are active and will move and wiggle in their corion using a plastic pasture pipette.
Transfer the 24 HPF embryos one at a time to individual wells of a 96 well plate. Using a micro pipette, remove any excess E three solution from the wells. Then using a multi-channel pipetter, add 200 microliters of E three solution to each well.
Next, add two microliters of DMSO to all the wells in columns one and 12. Then add two microliters of BCL prepared in DMSO as follows, one, micromolar to columns two and seven. Five micromolar to three and eight, 10 micromolar to four and nine 20.
Micromolar to columns five and 10. And finally 25 micromolar to column six and 11. Now cover the plate in aluminum foil and incubate it for six hours at 28 degrees Celsius.
Add 10 microliters of trica to each well to anesthetize the 30 HPF embryos. Then load the plate into molecular devices image express ultra open meta express software. Choose a configuration with a Forex objective and maximum pinhole diameter.
Configure the laser auto focus to detect the plate bottom and use a predetermined optimal Z offset. To detect the regions of interest, set the scan area as a single site of 2000 by 2000 pixels with no benning. Select the appropriate plate type and the 488 nanometer argon laser at excitation over emission wavelengths of 488 over 525 nanometers.
Then select a well containing a vehicle treated embryo and click find sample. The instrument will perform an auto-focus scan and acquire a single image to confirm proper image acquisition. Randomly select a vehicle treated well and acquire an image.
If the image is in focus and shows GFP expression in the head. Position the cursor over the brightest areas and verify that the fluorescence intensity is within the range of the 16 bit camera. Less than 65, 000 units.
Repeat these steps with another vehicle control and a few compound treated wells. If necessary, adjust laser power and gain such that brightest regions in the positive control images do not saturate. Next in the plate acquisition and control window, choose acquire plate to start plate scan.
The instrument will automatically acquire one image per well for the entire microplate and archive them in a SQL server database. Once the scan has finished, upload the images into D definition's. Developer by opening developer with the slinger option enabled.
Create a new workspace. Then from the file menu, select customized import. Next, navigate to the folder containing the images and open a sample file.
Choose an import filter that recognizes image format and file structure generated by the molecular devices image express ultra platform. Now to process the images, open a control image and load the desired cognition network technology or CNT algorithm or rule set. The custom algorithm used here is designed to automatically identify embryo, yolk and head and to quantify the brightness and area of GFP expressing head structures, execute the rule set to the stage of head and yolk detection.
To ensure proper assignment of yolk sack and head, modify the rule set to adjust the yolk detection threshold if the yolk is too large or too small. Next, execute the rule set to the stage of bright head structures detection. To ensure proper assignment of GFP expressing areas in the head, modify the rule set to adjust the head structures detection threshold as multiples of menial intensity until the detection of bright head structures matches visual observation.
Next, execute the entire rule set manually on several randomly chosen images generated from vehicle treated embryos and positive control images from PCI treated embryos. To run the algorithm on all plate images, select the plates to analyze and choose. Analyze, then navigate to the optimized rule set and analyze all wells.
Using the integrated job scheduler, empty wells or ambiguous images will automatically be excluded from the analysis during the scan. A continuously updating keep map is shown. Both the data and images are accessible during the run for visual inspection.
The numerical data and processed images are automatically saved. After all of the images have been analyzed, the data can be exported to third party software such as Excel, GraphPad, or prism. This figure shows archived scan images from a vehicle treated and A BCI treated well with and without CNT analysis.
Applied green fluorescent protein or GFP is detected in the eye, mid hind brain boundary and trigeminal ganglia of transgenic embryos. Note that both GFP intensity and the expression domains are expanded. In BCI treated embryos here, a dose dependent effect of BCI ON GFP reporter gene expression in transgenic embryos is seen.
The concentration required to elicit a half maximal response or EC 50 was approximately 12 micromolar. We've just showed you how to breed, collect and array transgenic zebra fish embryos into 96 well plates. We have added increasing concentration of PCI in own activator of the signaling pathway.
We have also shown how to automate the imaging of transgenic embryos and have analyzed the images with a custom designed algorithm based on cognition network technology. With this approach, we were able to measure quantitatively in a graded fashion the effects of a small molecule activator of fibroblast growth factor signaling. When doing this procedure, it's important to remember to carefully choose embryos to minimize variability between specimens.
That's it. Thanks for watching and good luck with your experiments.
