Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The overall goal of this procedure is to immunolocalize proteins that are activated in the zebrafish embryo. This method can help answer key questions in the developmental and cell biology field, such as the localization and activation of phosphorylated proteins in a whole embryo. The main advantage of this technique is that one can localize calcium dependent signaling in a whole embryo by immunostaining proteins activated by calcium.

After obtaining wild type or transgenic embryos according to the text protocol, add 0.003%PTU to the embryos in system water to block pigmentation and incubate to the desired developmental stage. When the embryos have reached the correct stage, add MESAB to the dish to anesthetize the fish. Once the fish are anesthetized, remove as much system water as possible, then add fresh 4%PFA and PBS and incubate at room temperature for three to four hours.

After the incubation, remove the PFA and use 100%methanol to replace it. Then, store the embryos at negative 20 degrees Celsius for at least 48 hours. To carry out immunostaining, place a minimum of 10 embryos for each experimental condition in labeled 1.5 millimeter micro-centrifuge tubes.

When the embryos have settled to the bottom of the tube, remove and discard the methanol and use progressive washes of decreasing concentrations of ethanol in PBTx to re-hydrate the embryos. Remove the last PBTx wash and add 0.5 milliliters of 10%normal goat serum or NGS in PBTx. Then, incubate at room temperature for at least one hour with gentle rocking.

Next, remove the solution and add 0.2 to 0.5 milliliters of a 1 in 5 dilution of mouse anti-acetylated tubulin monoclonal antibody and 10%NGS and PBTx. Incubate the embryos with gentle rocking at room temperature overnight. In the morning, remove and discard the antibody and perform three washes using 0.5 milliliters of 2%NGS and PBTx with gentle rocking for five minutes each.

Dim the overhead lights and add 500 microliters to each sample of a 1 in 500 dilution of flourescently conjugated secondary antibody in 10%NGS and PBTx. Incubate in the dark at room temperature for four hours with gentle rocking. After the incubation, wash the embryos three times.

To co-immunostain, add a 1 in 20 dilution of anti-phospho CaMKII antibody. Incubate in the dark with gentle rocking overnight. The next morning, after washing the embryos with 2%NGS and PBTx three times, add a 1 in 5 dilution of red 568 fluorescent conjugated goat anti-rabbit IgG and 10%NGS and PBTx.

After incubating in the dark at room temperature for four hours, wash the embryos three times and store in PBTx or 50%glycerol in PBS depending on the imaging procedure. To mount the embryos for imaging, place one to five embryos on a glass slide. Use four number one stacked cover slip fragments on either side of the fish to create a chamber.

And place a cover slip on top. With the 100X oil immersion lens and transmitted light bring a single embryo into focus. Turn off the light.

Turn on the confocal microscope with the appropriate lasers and engage the remote focus. Open the confocal program and in the acquire bar, select the proper objective. Then, in the XY basic bar, click on the 1024 button to set the image size.

In the laser and detector bar, click on the red 488 and green 568 boxes to turn on the laser detector for each channel and set the pinhole to medium. Then, in the gain bar, adjust the gain for each channel to visualize it. Next, in the acquire settings bar, click live to begin acquiring images.

In the view settings bar, uncheck the force integral zoom box to center in the live window. In the acquire settings bar under the Z tab, step through the layers of the image. After selecting the number of layers to incorporate into the image, move to some point in the center of the Z plane.

Under the Z tab, click the small red reference box. This will zero the RFA at the point chosen as the center. In the box labeled step size, select the thickness of the layers.

Pay attention to the file size box and try to limit the files to one gigabyte. To find the top extreme of the image, click the circle next to top and move through the Z layers. Now click the circle next to bottom and the computer will take the image back to the layer as center.

Then, in the acquire settings box, click on live again to stop the laser acquisition. In this panel, click the red boxes labeled average and Z stack. These boxes will turn green.

In the acquire settings box, click single to acquire the entire series of images. Monitor the progress as it scans in both channels and through the entire Z stack. To save, click on the volume window and click save as and name the file.

To volume render the file, while the Z stack is still open, select the data pulldown menu and click on volume render. Save the rendered file as a dot tif file. This figure shows immunostaining for P-CaMKII and acetylated tubulin, which is a standard marker for cilia in the zebrafish Kuppfer's vesicle or KV.Feeding primary cilia generate a circular flow of fluid, which leads to an elevation of calcium in the ciliated cells lining the KV and the activation of CaMKII in discrete locations as seen here.

These figures show P-CaMKII as it appears on the apical surface of ciliated cells lining specific regions of the perinephric ducts between 24 and 72 hours post-fertilization or HPF. At one day of development, the zebrafish embryonic ear fixed with PFA and methanol retains its structures and allows the detection of CaMKII staining at the base and along the length of the kinocilum. At 72 HPF, the inner ear was counterstained with Alexa 488 phalloidin and anti-acetylated tubulin with Alexa 568.

The PFA methanol fixation preserved both F-actin and tubulin. This 30 HPF fish expressed a membrane-targeted GFP, which would be lost with methanol fixation. PFA fixation resulted in a diminished P-CaMKII signal compared to fixation with PFA and methanol.

However, at this stage of development, the signal is strong enough to be detected without methanol. Once mastered, this technique can be done in two days if it is performed properly. While attempting this procedure, it's important to remember to use fresh embryos and fresh free agents.

After watching this video, you should have a good understanding of how to localize phosphoproteins in zebrafish embryos.