Our protocol describes the use of a hydrogen peroxide biosensor in cultured zebrafish neurons and larvae. This protocol will help researchers to dissect the role of reactive oxygen species in normal physiology and pathophysiology. The main advantage of this technique is the real time detection of hydrogen peroxide in living animals and cultured cells.
This method can be applied to other animals, like rodent model system. To prepare the coverslips, use acid-cleaned coverslips stored in 100%ethanol. Use forceps to remove one coverslip from the storage container, and flame it to remove residual ethanol.
Air dry the coverslip completely by placing it at an angle inside a 35 millimeter culture dish. Prepare Poly-D-Lysine or PDL, or working solution. Apply PDL to the center of each coverslip, avoiding spreading of the solution to the edges and incubate the PDL for 20 to 30 minutes at room temperature.
Make sure the PDL does not dry out. Wash the PDL with 0.5 milliliters of sterile water and let the plates dry completely. Prepare laminin working solution.
And apply it to the center of each coverslip, making sure to avoid the spread of solution to the edges. Incubate the plates at 37 degrees Celsius in a humidified incubator for two to six hours, avoid a drying of the laminin solution. To perform a embryo dissection in plate retinal ganglion cells or RGCs, prepare and label four 35 millimeter tissue culture dishes and fill them with four milliliters of 70%ethanol.
E2 media one. E2 media two. And E2 media three, on the day of dissection.
Keep the dishes in the fridge. When zebrafish embryos are 34 hours post fertilization, take the culture dishes coated with laminin out of the incubator. And wash the cover slips three times with 0.5 milliliters of 1X PBS.
After the final wash, add four milliliters of ZFCM+media to each culture dish. Avoid drying the plate. Retrieve the prepared refrigerated culture dishes and let them equilibrate to room temperature.
Fill four to six PCR tubes with 15 microliters of ZFCM+media. Retrieve zebrafish embryos from the incubator and immerse embryos in a 35 millimeter tissue culture dish containing 70%ethanol for 5-10 seconds to sterilize it. Using a transfer of pipette, transfer embryos to the E2 media one dish, containing sterile E2 media to wash off excess ethanol.
Then, transfer the embryos to the E2 media two dish and remove their chorions with sharp forceps. Finally, transfer embryos to the E2 media three dish to perform dissections. Using a pair of fine forceps, position and hold embryos anterior to the yolk with one of the forceps.
And remove the tail posterior to the yolk sac with the other forceps. Grab the neck with forceps and take off the head to expose the brain and eyes to the E2 media. With the tip of fine forceps, gently roll the eyes from the head while holding the cranial tissue down with a second forceps.
Transfer four eyes to one of the previously prepared tubes containing ZFCM+Gently triturate up and down with a p20 pipette about 45 times to dissociate cells. Transfer the ZFCM+with the dissociated cells to the center of the coverslips. Maintain cultures on the bench top at 22 degrees Celsius on a polystyrene foam rack to absorb vibrations.
On the day of imaging, check cells under the microscope to validate growth of RGC axons. For live cell imaging, transfer the coverslips from the culture dish to a live cell imaging chamber. Use an inverted microscope equipped with a DIC objective OG-590 Longpass red filter and an EMCCD camera.
Before imaging, replace the ZFCM+medium with ZFCM-After positioning the cells with the 10X objective, acquire images at 60X magnification using an oil immersion objective. Use an additional 1.5X magnification. First acquire DIC images.
Then, image roGFP2-Orp1, using the appropriate filter set. After taking the first set of images, exchange media with media containing different treatment solutions. Media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.
For in vivo imaging, keep 22 to 24-hour post-fertilization embryos in E3 media, containing 0.003%phenyl thiourea without methylene blue. Exchange media and remove dead embryos daily. At the desired age, anesthetize and mount embryos in 1%agarose on 35 millimeter glass bottom culture dishes.
Embryos can be oriented dorsally, ventrally, or laterally, depending on the region of interest for imaging. After the agarose solidifies, fill the dishes with E3 media without methylene blue, but with 0.016%tricaine. Set up the microscope for imaging.
Use an inverted laser scanning confocal microscope to image embryos mounted on the bottom of an agarose drop. Excite roGFP2-Orp1, and acquire corresponding images with the desired emission filters. Acquire z-stacks with five micro meter section thickness through the desired part of the embryos.
After imaging, remove embryos from Agarose with fine forceps and keep them in the incubator and methylene blue free media with phenyl thiourea until the desired age. A representative image of the hydrogen peroxide biosensor and cultured zebrafish RGC extended axons is shown here. The cell body, axon, and growth cones are clearly visible in individual neurons.
The addition of hydrogen peroxide to the culture media increases the ratio values, showing that real-time changes can be detected with this system. Quantification of hydrogen peroxide levels is shown in panel B.To determine hydrogen peroxide levels in whole zebrafish embryos, the mRNA was injected during the one cell stage, causing all tissues to express the roGFP2-Orp1 biosensor. The head region of zebrafish larvae is shown at two different time points, focusing on the hydrogen peroxide levels in the retina.
The basal levels of hydrogen peroxide in the zebrafish embryos at two days post-fertilization and five days post-fertilization were measured. At two days post-fertilization, the ratio values were significantly lower than at five days post-fertilization. Furthermore, each animal showed a different level of increase in their retinal hydrogen peroxide content.
Using fine forceps for removing the eyes and gentle dissociation of the eyes with the pipette are the most critical steps in this procedure. This protocol can be followed by drug treatments to investigate the factors involved in ROS signaling.
