The overall goal of this procedure is to analyze whole zebrafish embryos between the four cell stage and 32 hours post fertilization for the presence and location of apoptotic cells. This is accomplished by first coating embryos at a desired time point. Next, the embryos are fixed and permeable.
Then the embryos are incubated with rabbit anti activated caspase three antibody, followed by a fluorescently tagged anti rabbit secondary antibody to fluorescently label apoptotic cells. Finally, the embryos are analyzed by fluorescence microscopy. Ultimately, results can be obtained that show differences in number and location of apoptotic cells between control and treated or mutant embryos through immunofluorescence.
The main advantage of this technique over existing methods like analysis of apoptotic cells by Aine orange staining or the tunnel assay, is that the Caspase three assay is a highly reproducible and relatively inexpensive method for experiments in which photographic documentation and or quantitation of apoptotic cells is required across multiple samples. Demonstrating the procedure will be Shelly SOLs, a graduate student from my laboratory and Christian Tono, a technician from my laboratory Under a chemical fume hood. Prepare 50 milliliters of 4%para formaldehyde or PFA by adding PFA powder, the one XPBS shield from light and stir at 60 degrees Celsius for one hour to dissolve store at four degrees Celsius for one month or at minus 20 degrees Celsius for two years.
Prepare 100 milliliters of 4%Methylcellulose by heating 30 milliliters of water to 80 degrees Celsius. Then add four grams of Methylcellulose and stir to dissolve at room temperature. Four hours to overnight.
Bring the volume up to 100 milliliters and mix at four degrees Celsius for one hour. Store at room temperature, collect embryos immediately after fertilization and let them develop until the desired time point between the four cell stage and 32 hours post fertilization or HPF four embryos between tail bud stage and 32 HPF. Remove the corion by incubating embryos in 100 microliters of 10 milligrams per milliliter.
Pronase in 10 milliliters of one x egg water for 30 minutes. At room temperature. Pour off the pronase solution slowly.
Add one x egg water and repeat the rinse two more times. Then transfer up to 40 embryos to a 1.5 milliliter tube and use one milliliter of one X-P-B-S-T to rinse two times after removing all but about 100 microliters of the PBT. Add one milliliter of 4%PFA.
Lay the tubes on their side in an appropriate size container for ease of transport and place on a gentle rocker at four degrees Celsius overnight or up to a week after fixing the embryos. Remove most of the PFA solution and carefully in a dropwise fashion, add one milliliter of ice cold 100%methanol. Lay the tubes on their sides and place at minus 20 degrees Celsius for two hours.
Alternatively, embryos can be left at minus 20 degrees Celsius for up to 24 hours. After the incubation, allow the embryos to settle to the bottom of the tube before removing most of the methanol and carefully adding one milliliter of one XPBS with 0.3%trite x and 1%DMSO. Immediately remove the PDT and replace with fresh PDT.
Then lay the tubes on their sides and rock for 30 minutes at room temperature. After carefully removing most of the PDT add 500 microliters of blocking buffer and ensuring that the embryo stays emerged in solution Gently rock for one hour at room temperature. Next, add one microliter of rabbit anti activated caspase three antibody and additional non rabid antibodies.
If desired, gently rock the embryos for two hours at room temperature or overnight at four degrees Celsius. After removing the antibody solution, add one milliliter of PDT to quickly rinse the embryos. Then replace with fresh PDT and gently rock for 30 minutes at room temperature.
Before repeating the wash, remove the PDT wash and repeat the blocking step. Then add the appropriate Fluor four conjugated secondary antibodies. Shield from light and gently rock for one hour at room temperature under a fluorescent microscope with the appropriate filter.
Analyze the embryos in one XPDT. If positive controls have bright fluorescence with minimal background, then transfer them to a 3.5 centimeter Petri dish with a thin layer of 4%Methylcellulose or a depression slide with a large drop of methylcellulose using an angled probe. Orient an embryo of interest for imaging taking images that represent fluorescence intensity and patterns observed in the majority of the embryos from that sample.
This figure demonstrates that injection of embryos with high concentrations of mRNA encoding potent proto BH three only. Proteins such as BIM or NOA quickly gives rise to active caspase three. By the four cell stage of development, embryonic death subsequently occurs within one hour.
At later stages of development, fluorescence intensity is more robust. For example, shown here are bright active caspase three positive cells in sphere stage embryos that have been injected with moderately toxic levels of mRNA encoding the pro apoptotic BH three only. Gene Puma messenger, RNA, encoding the antia potto BCL two gene was co injected to prove that injection of Puma mRNA induces apoptosis through the intrinsic mitochondria mediated pathway.
IR induced apoptosis and zebrafish requires P 53 mediated transcription of BH three only genes such as puma. The delay in IR induced apoptosis shown here is consistent with the requirement for P 53 mediated transcription. The caspase three protocol should give rise to a robust signal to noise ratio.
This figure compares the optimal caspase three assay result to two common suboptimal results that arise when critical steps are compromised as outlined in the discussion section of the text protocol. First is a weak signal to noise ratio due to background fluorescence in animal tissue, and second is yolk fluorescence that distracts from the signal of interest. Finally, as shown here, co labeling experiments using transgenic GFP expressing lines, approximate tissues undergoing apoptosis, the transgenic OLE two EGFP VU, 12 line labels, motor neurons and oligodendrocytes, and the transgenic elev L three EEG FP line labels, all differentiating neurons.
The results suggest that both neurons and oligodendrocytes are sensitive to IR induced apoptosis Following this procedure. Other methods like cos staining with tissue specific primary antibodies and embryo suctioning can be performed in order to pinpoint the exact identity of cells undergoing apoptosis.
