Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

1. Preparation of Solutions Prepare 500 ml of 1x PBST (1x PBS, 0.1% Tween-20), and filter sterilize. 1x PBST can be kept at room temperature (RT) indefinitely as long as there are no signs of bacterial growth. CAUTION: Prepare 50 ml of 4% paraformaldehyde (PFA) in a chemical hood by dissolving powdered PFA in 1x PBS at 60 °C for 1 hr and covering in aluminum foil to protect from light. Store at 4 °C for 1 month, or at -20 °C for 2 years. Prepare 500 ml of fresh 1x PDT (1x PBST, 0.3% Triton-X, 1% DMSO). 1x PDT can be stored at 4 °C for up to one month. Prepare 50 ml of blocking buffer (1x PBST, 10% heat-inactivated fetal bovine serum, 2% bovine serum albumin). Create 10 ml aliquots, and store at -20 °C for up to 1 year. Once thawed, blocking buffer can be stored at 4 °C for up to 48 hr. Prepare 100 ml of 4% methylcellulose by heating 30 ml of water to 80 °C. Add 4 g of methylcellulose to the hot water, and mix using a stir bar on a room-temperature stir plate.  Thorough mixing usually takes between 4 hr to overnight. Add the remainder of (unheated) water, and mix at 4 °C for 1 hr. Store at RT. If using embryos between tailbud stage and 32 hpf, prepare 50 ml of 10 mg/ml pronase, and store for up to 1 week at 4 °C. Otherwise, store in 10 ml aliquots at -20 °C for up to 1 year. Place 50 ml of 100% methanol at -20 °C. Prepare 60x egg water by dissolving 280 g Instant Ocean Sea Salt in 2 L of distilled water (as in Westerfield16). Prepare 1x egg water by diluting the 60x stock in deionized water. 2. Preparation of Embryos Collect zebrafish embryos in a Petri dish in 1x egg water between the 4-cell stage and 32 hpf. For embryos between the 4-cell and tailbud stages, proceed to step 2.2. For embryos between tailbud stage and 32 hpf, remove chorions by adding 100 μl of 10 mg/ml pronase to embryos in 10 ml of 1x egg water. Note: Pronase activity can be variable between batches. Thus, final pronase concentration should be optimized for dechorionation when new batches are obtained. Let incubate at room temperature for 30 min. Pour off egg water / pronase solution into waste, and slowly add back fresh 1x egg water. Rinse 2x by pouring off existing egg water (and free-floating chorions), and gently add new egg water. Note: Most chorions should be removed at this point. With a transfer pipet, gently suck embryos up and down until remaining chorions are removed. Transfer up to 40 embryos to a 1.5 ml tube. Rinse 2 times with 1 ml of 1x PBST.   Remove PBST, leaving liquid just above embryos (approximately 100 μl volume should remain in the tube). Add 1 ml of 4% PFA. Lay tubes on their side (so embryos spread out along length of tube) in an appropriate-size container for ease of transport (e.g. a Petri dish or a pipet box top), and place on a gentle rocker at 4 °C overnight or up to a week.   For embryos between the 4-cell and tailbud stages, manually dechorionate the fixed embryos with forceps. 3. Immunofluorescence Procedure Place tubes in a tube rack so that all embryos sink to the bottom of the tube. Remove PFA (to appropriate waste container) leaving liquid just above level of embryos. Following fixation (step 2.3), embryos should never be exposed to air in order to prevent background antibody staining. Add 1 ml of ice-cold 100% methanol (to permeabilize) drop-wise. Embryos are now fragile after the fixation, and extra care must be taken to prevent them from tumbling within the tube during all subsequent removal/addition of liquids. Lay tubes on their sides, and place at -20 °C for 2 hr. Alternatively, embryos can be left at -20 °C for up to 24 hr. Place tubes in a rack to collect embryos at the bottom of the tube. Remove methanol, leaving liquid just above level of embryos. Carefully add 1 ml 1x PDT. Remove PDT, and add 1 ml of fresh 1x PDT. Lay tubes on their sides and gently rock at RT for 30 mins. Remove PDT, and repeat 30 min wash with fresh PDT. Remove PDT until liquid is just above the level of the embryos, and add 500 μl blocking buffer. Lay embryos on their side in a transport container, and ensure that embryos do not travel more than 2/3 up the length of the tube. Otherwise, embryos get caught in the lid of the tube and outside of the liquid.   Carefully place transport container with tubes on a very-gentle rocker at RT for 1 hr. Stand tubes upright in a tube rack, and open all tubes to check for embryos stuck on the lid. For any tubes with embryos on the lid, close the top, and gently rock the tube by hand to bring all embryos back into the liquid. Add 1 μl rabbit anti-activated Caspase 3 antibody (1:500 dilution). If performing a colabeling experiment, simply add the appropriate dilution of the second nonrabbit-derived antibody in addition to the anti-activated Caspase 3 antibody. Carefully lay embryos on their side, and gently rock at room temperature for 2 hr. Alternatively, embryos can be incubated at 4 °C overnight. Stand tubes in a tube rack. Remove block/antibody solution. Add 1 ml of 1x PDT. Be sure to invert tube by hand to collect any embryos stuck on the lid of the tube. Then, open all tubes to ensure all embryos have been removed from the lid. Repeat step 3.4. Repeat steps 3.5-3.10, but substitute the anti-activated Caspase 3 antibody with the appropriate dilution of an anti-rabbit fluorophore-conjugated antibody that absorbs blue light and emits green light. However, any fluorophore-conjugated antibody with a user-preferable absorption/emission spectra can be used here. After addition of the secondary antibody, it is important to keep embryos in the dark for the remainder of the assay. Cover the transport container with aluminum foil during the incubation steps, but do not worry about light exposure when changing buffers. If performing a colabeling experiment, simply add the appropriate dilution of a complementary fluorophore-conjugated secondary antibody at this step. Note: If the user wishes to colabel active-Caspase 3 with Gfp-expressing cells, then the rabbit anti-active Caspase 3 antibody should be followed here by a nongreen-fluorescing anti-rabbit secondary antibody, and the anti-GFP antibody should be followed by a green-fluorescing nonrabbit secondary antibody. This will eliminate confusion if some Gfp fluorescence remains visible after fixation of embryos. 4. Analysis of Apoptotic Cells by Fluorescence Microscopy Analyze embryos in 1x PDT in a small Petri dish or glass spot plate under a fluorescent microscope using the appropriate light filter. If positive controls have bright fluorescence with minimal background, then proceed to step 4.2. (If not, consult the troubleshooting section in the Discussion, and repeat the protocol with new embryos.) Embryos can be stored at 4 °C in 1x PDT for up to 48 hr. Pour 4% methylcellulose in a 3.5 cm Petri dish, just enough to cover the bottom of the dish. Alternatively, a glass spot plate or depression slide can be used with a large drop of methylcellulose in the depression. Carefully suck embryos from the bottom of the tube into a clean transfer pipet and transfer to the Petri plate for visualization. Orient an embryo of interest for documentation using an angled probe. There will likely be some variability between embryos from the same sample, so it is important to take images that represent fluorescence intensity and pattern observed in the majority of the embryos from that sample. Documentation should be performed within 48 hr of completing the assay to prevent accumulation of non-specific background fluorescence. Residual 4% methylcellulose will not interfere with subsequent PCR reactions required for genotyping of embryos. Note: To aid in immobilization of an embryo at a certain angle, it is helpful to gently push the embryo to the bottom of the dish so that the yolk or some other part of the embryo (depending on the desired view) is resting on the bottom of the dish. Then, use the probe to move either the embryo or the immediately surrounding methylcellulose (while simultaneously visualizing under the microscope) to obtain the desired angle for documentation.