The overall goal of this procedure is to automatically detect estrogen responsive cells in live zebrafish larvae. This is accomplished by first breeding five X-E-R-E-G-F-P adult fish to generate estrogen reporter zebrafish embryos. Next, the embryos are placed in individual wells of a 96 well plate.
Then estrogens are added to the plate of embryos. Finally, the plate is scanned for fluorescence using an epi fluorescent microscope with a motorized stage. Ultimately, fluorescent microscopy is used to show tissue specific activity of estrogen receptors.
So the main advantage of using this technique over other traditional techniques like high throughput or high content screening, is that tissue specific fluorescence in zebrafish larvae can be imaged using just a standard epi fluorescent microscope. Equipped with a motorized stage, no specialized plate reader is necessary. Caution chemicals with BPA and estradiol are hazardous and can have deleterious effects on the human fetus.
Handle endocrine disruptors with care and always use proper personal protective equipment. In this demonstration, zebrafish will be exposed to the estrogens 17 beta estradiol, bisphenol A and genine in various doses, beginning at 48 hours post fertilization after breeding zebrafish, and collecting and equating eggs. According to the text on the day of treatment, dilute thaw stock chemical solutions, one to 1000 in one milliliter of E three B plus PTU vortex vigorously.
As a vehicle control. Use a one to 1000 dilution of DMSO in E three B and PTU using a large bore plastic transfer. Pipette transfer three embryos per well into a 96 well plate preparing three wells per condition.
Avoid placing embryos in outer wells to prevent evaporation of medium during prolonged treatment. Working quickly to avoid drying out the embryos, remove the medium from each well and add 200 microliters of well mixed treatment solution. Visually confirm that all embryos are submerged in the solution and are not stuck to the side of the well.
Incubate the embryos at 28.5 degrees Celsius at 72 hours. Post fertilization anesthetize embryos by adding 10 microliters of four milligrams per milliliter of trica. To each, well place the 96 well plate onto a motorized stage and using the Zeiss Zen Blue 2011 software.
Calibrate the stage by selecting the sample carrier option and choosing multi well 96. Then select calibrate before following the stepwise directions, using a 10 x objective and the bright field or BF setting, identify a positive control embryo and set the exposure settings appropriately for BF and GFP channels, making sure that fluorescence signal is not saturating the camera. Focus on a single embryo and program the microscope to acquire a total of three Z sections that include one image 50 micrometers above and one 50 micrometers below the current focal plane.
Next, to set the software parameters to acquire a tiled image using GFP and BF channels. Under the acquisition tab, select advanced setup, select tile regions, then tiles. Choose the circle.
Well option select carrier and fill factor 90%to capture multiple images of the selected wells, such that 90%of the area of each well will be visible in the composite image. Under options, select the amount of overlap desired, such as five to 10%which allows for a smooth seam between images. Then start imaging to manually capture images using a 20 x objective and the BF setting, identify a positive control embryo.
Set the BF and GFP settings as before and take images. This figure shows composite images from individual wells of a 96 well plate. Each image is composed of 59 individual images with 5%image overlap.
Note that the live zebrafish are oriented randomly within each well. Yet we are able to distinguish fluorescence in the heart from fluorescence in the liver. Brightfield images are useful as references to assess zebrafish orientation and visualize morphological abnormalities.
The images shown here were captured with a 20 x long working distance objective for more detailed analysis, and were taken directly after the automated scan just shown without removing the plate from the microscope stage. Using a 20 x objective, the fluorescence in the heart can be precisely localized to the atrial ventricular valves pointed out by the arrows and the aortic valves as indicated by the arrowhead. Once this technique is mastered, images from 3 96 wall plates can be captured per day.
Don't forget that working with endocrine disrupting compounds can be hazardous, so always take the proper precautions such as wearing the proper personal protective equipment while performing this procedure.
