The overall goal of the following experiment is to qualitatively and quantitatively measure oxidative stress in living zebra fish embryos. This is achieved by adding an oxidative solution to prepared embryos for oxidative stress induction, or by creating a wound margin at the embryo's tail fin. The embryos are then processed for oxidative stress detection by either a single cell method or by a whole mount method.
Next, the preparations are incubated with a Ross detecting probe. Results can show the amount of oxidative stress and Ross level based on confocal microscopy of hole mounts or cyto fluoro metric analysis of single cells. The main advantage of this technique over existing methods like immuno Fluor for oxy stress markers is that it allows the raw detection in the context of a living organism and quantification at the level of single tissues.
This method can help answer key questions in the basic research field, such as exploring oxidative stress conditions in the context of a pathological state. Individuals new to the admitted may struggle because of accidental tissue damage because the raw sensitive molecular probes may be unplanned source of acidity. And because high levels of oxid stress must result in cell death, while too little may be difficult to detect with practice and experimentation, these issues will only result themselves When preparing the solution for inducing oxidative stress to mitochondria composed of rot known in DMSO, it should be made with rot known stock at a concentration between five and 50 micromolar and never above 100 micromolar rotten known is toxic and hazardous.
Take heed of the labeled precautions. The probe solution for hole Mount Ross detection should not be exposed to any light or oxygen during its preparation, and it must be prepared fresh for the single cell Ross detection method. A stop solution of FBS in PBS must be prepared and kept at four degrees Celsius.
Other preparations include setting the zebrafish air incubator to 28 degrees Celsius and for the single cell method cooling the centrifuge to four degrees Celsius. Zebrafish crosses embryo collection and anesthesia are all performed. Using standard methodology, collect embryos of interest between 48 and 72 hours post fertilization.
After anesthetizing the embryos wash out the anesthetic twice, then load the embryos into three dishes with no more than 30 per dish for single cell raw detection. Collect at least 35 embryos per condition in multiple dishes. Next, apply 10 milliliters of oxidant solution or as a negative control.
Apply 10 milliliters of water, then wait 10 to 60 minutes while the embryos incubate at 28 degrees Celsius. Also prewarm HBSS to 28 degrees Celsius for the wash. After the incubation, transfer the embryos to a new dish of warm HBSS and swirl.
Then proceed with either whole mount or single cell Ross detection. A more physiological option is to generate stress after tissue damage by using the tailfin wounding technique described by NI Tomer and company. After oxidant treatment, transfer groups of up to 10 embryos to small tubes.
Rinse them heavily with HBSS and protect them from light with foil. Next, add a milliliter of raw detection solution to each tube and incubate the tubes for 15 minutes at 28 degrees Celsius while they incubate. Prepare a glass slide to contain both the control and experimental condition.
Cover a depression slide with 300 microliters of methylcellulose. Do not make bubbles when ejecting the methylcellulose. After the embryo incubation, quickly replace the solution in the tubes with two milliliters of HBSS and invert the tubes several times to wash the embryos.
Then aspirate the embryos into the tip of a micro pipette and gently eject them into the treated slide. Orient the embryos using a fine nylon line and proceed with using a fluorescent or confocal scanning microscope for analysis. Be sure to analyze all the embryos under the same settings.
Transfer the embryos from the HBSS wash to a new dish, removing as much of the solution as possible. Now manually dec coate the embryos. This is truly necessary to dissociate embryos into single cells.
Next, move the embryos into a 24 well plate load 15 embryos into each. Well optimally fill three wells for each condition. Next, remove all the solution and replace it with 300 microliters of HBSS 30 microliters of collagenase p and 50 microliters of trypsin EDTA.
Using a one milliliter pipette tip, homogenize the embryos with trier. Once all the wells are prepared, transfer the plate to 28 degrees Celsius for at least 20 minutes every five minutes, tritrate the samples to ensure good homogenization. Also put some micro fuge tubes on ice to collect the tissues when they are ready.
At 20 minutes, take a sample and check that the tissue is disrupted into a single cell suspension under a microscope. If not, continue the incubation for up to 30 minutes in total. When the tissue is ready, stop the reaction with an additional 200 microliters of stop solution.
Mix this in with TRI using a one milliliter pipette tip. Next, transfer the content of each well into a pre chilled micro fuge tube and keep these tubes on ice out of the light. Then centrifuge the samples at 250 GS for five minutes at four degrees Celsius.
And then remove the supernatant by aspiration. Resuspend the cell pellet in ice cold HBSS with gentle tri, and check that the suspension has at least 2 million cells. Next, resuspend the cells in a milliliter of the raw sensitive probe solution.
Incubate them at room temperature and in the dark for about three minutes. Adjust this incubation time empirically. Next, transfer the cells to fax tubes shielded from light.
Proceed with fax analyses to detect the transgenic marker. Fluorescence and Ross probe fluorescence During your analysis. Always calculate loss levels as fold increases compared to control samples in order to normalize the background fluorescence.
All Mount Ross detection was used to examine hydrogen peroxide accumulation at a wound site. Intact and injured tails were compared after 20 minutes of oxidant treatment. Non-specific signal was detected in both conditions.
The same experiment with the pre-treatment of the embryos to lower hydrogen peroxide levels. Using OX inhibitor VAs 28 70 revealed that the detected signals were indeed dependent on Ross accumulation rot. Known treated embryos were also examined by whole mount.
Under high magnification anatomical regions could be identified. That produced Ross as indicated by fluorescence counting of Ross positive cells by fax was performed for single cell Ross detection. This analysis was carried out without the inclusion of dead cells.Control.
Cells without treatment were also analyzed. Comparing these results made quantification of Ross accumulation very easy. The assay proves to be excellent for testing any number of experimental manipulations.
Once mastered this, a technique can be done in 90 minutes if it is performed properly. Don't forget that working with molecular probes could be extremely hazardous and the precursion. Such use of gloves should always be taken while performing this procedure.
