The overall goal of the following protocol is to measure the metabolic profile of zebrafish embryos in response to specific genetic or physiologic alterations. This is achieved by first modulating a particular genetic or physiological pathway using a selective pharmacologic compound. Next, when the desired developmental stages reached basal respiration is measured in control and treated embryos using the seahorse XF 24 analyzer.
Then respiration parameters are calculated from the data results are obtained that reveal that metabolic profiles can be studied in the developing zebrafish in vivo, and that these profiles can be modulated by targeting a specific gene during embryonic development. The main advantage of cyte in regards with already available techniques like cell-based assay or indirect measurement of respiration, is that we are dealing with the whole organism, a whole embryo, allowing us to look at all the aspects of mitochondrial respiration. This technique can help answer key questions in metabolism by measuring whole animal respiration and mitochondrial function.
To begin treatment, collect zebrafish embryos after fertilization for in C two hybridization or oil red O staining at 0.2 millimolar one, PHENYL two, theo uia. To prevent pigment formation, incubate the embryos for about 26 hours. Add the desired chemical at an appropriate final concentration with a vehicle only control before using the seahorse XF 24 analyzer, run the calibration that houses the oxygen and hydrogen fluoro force to prepare the 24 well XF 24 islet plate.
Begin by adding 700 microliters of E three medium to each. Well then add one embryo to 20 of the wells to ensure that the specimen remains in the measurement chamber. Throughout the assay, apply an eyelet capture screen to the top of each well.
10 of the wells are used for control Embryos indicated with a C and 10 of the wells are used for treated embryos indicated with a T.The black boxes indicate the wells without embryos, which are used to control for temperature fluctuations across the plate. Now load the plate into the seahorse analyzer. To measure respiration, use the basal respiration maximum respiration program to establish basal respiration.
Measure three times for two minutes each waiting one minute between each measure next at the mitochondrial proton four on coupler FCCP at a final concentration of 2.5 micromolar. Repeat the measurement cycle five to eight times after establishing basal respiration. Use the A TP turnover Proton leak program to measure a TP turnover at 25 micromolar of all liga mycin and inhibitor of a TP synthase and run the measurement cycle eight times.
Then use the resulting respiration values to calculate the difference between a liga and mediated respiration and basal respiration. Repeat this process by adding 25 micromolar of rotenone, a complex one inhibitor. Then determine uncoupled respiration by calculating the difference between olim and mediated respiration and respiration following the addition of roton when the program is complete.
The seahorse analyzer calculates the mean values of the 10 control and 10 chemically treated embryos to perform oil. Red o staining to analyze lipid deposition. When the treated and untreated embryos reach 50 hours post fertilization, use fine forceps to coate them and fix them in 4%Paraform aldehyde in PBS overnight at four degrees Celsius.
Wash the embryos once in PBT for five minutes, then incubate them in 60%Two propanol for one hour, followed by two hours in 0.3%oil red O and 60%Two propanol rinse the embryos briefly in 60%Two propanol two times followed by PBT once finally transfer the embryos from 30 to 50, then to 75%glycerol for photography as shown here. Treatment of embryos with a specific enzyme inhibitor led to about a twofold increase in respiration, which was associated with an increase in lipid deposition, especially in the cephalon and underneath the otic vesicle. While attempting this procedure, it is important to work with embryos at the same develop stage.
This is particularly true when you're comparing mitochondrial respiration between a control embryo and a mutant or a pharmacologically treated embryo that could be a bit delayed in development. So the development of this technique has allowed researchers in the field of metabolism to explore whole animal respiration and mitochondrial function using zebra fish as a vertebrate model.
