Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

The overall goal of this protocol is to label and visualize multiciliated cells of the zebrafish in situ. This method can help answer key questions in the developmental biology field such as what factors regulate multiciliated sulfates in the embryonic zebrafish kidney? The main advantage of this technique is that both protein and RNA transcripts can be labeled simultaneously which is useful in the absence of zebrafish-specific antibodies.

We first had the idea for this method when we went to label multiciliated cells in the pronephros while analyzing the presence of cilia after making changes to the embryo. Begin by fixing the zebrafish embryos at 24 hours post fertilization in five milliliters of 4%paraformaldehyde for two to four hours at room temperature without agitation. Next, wash the embryos two times in PBS with 0.1%Tween 20 or PBST followed by one wash with five milliliters of 100%methanol.

Then, incubate the embryos in five milliliters of fresh 100%ethanol for at least 20 minutes at minus 20 degrees Celsius. To prepare the embryos for hybridization, rehydrate the neonates in five milliliters of 50%methanol and PBST for five minutes followed by five minutes in five milliliters of 30%methanol and PBST. Wash the embryos two times for five minutes each in five milliliters of PBST and incubate the embryos in five milliliters of a one-to-1, 000 proteinase K and PBST solution for two minutes immediately followed by two more washes in five milliliters of PBST.

Fix the embryos in five milliliters of 4%paraformaldehyde for at least 20 minutes at room temperature followed by two washes in five milliliters of PBST 20. To facilitate interactions between the embryos and the hybridization solution, transfer the embryos into five milliliters of PBST in upright flat-bottom micro centrifuge tubes in a micro centrifuge rack and wash the embryos two times for five minutes each in 1.5 milliliters of hybridization solution. After the second wash, incubate the embryos in 1.5 milliliters of fresh hybridization solution at 70 degrees Celsius in a hybridization oven for four to six hours.

Then, replace the hybridization solution with 10 microliters of RNA probe of interest and 500 microliters of hybridization solution for an overnight probe hybridization at 70 degrees Celsius. The next morning, wash the embryos two times in 1.5 milliliters of 50%formamide and 2X saline-sodium citrate buffer or SSC for 20 to 30 minutes per wash at 70 degrees Celsius. Then, wash the embryos one time in 1.5 milliliter of 2X SSC alone at 70 degrees Celsius for 15 minutes followed by two washes in 1.5 milliliters of 0.2X SSC at 70 degrees Celsius for 20 to 30 minutes per wash.

After the second wash, replace the 0.2X SSC with 1.5 milliliters of blocking reagent for an overnight incubation at four degrees Celsius. The next morning, replace the blocking reagent with 0.2 milliliters of anti-digoxin in horseradish peroxidase and blocking reagent at a one-to-1, 000 ratio for a three-hour incubation at room temperature protected from light. Then, wash the embryos two to four times in 1.5 milliliters of malic acid buffer for 10 to 15 minutes per wash followed by an overnight incubation at four degrees Celsius in 1.5 milliliters of fresh malic acid buffer.

The next morning, replace the malic acid buffer with 1.5 milliliters of PBS and wash the embryos two times in 1.5 milliliters of PBS for five minutes per wash. Incubate the embryos in 0.2 milliliters of Si3 fluorescent standing solution for 60 minutes followed by four consecutive 10-minute washes in 1.5 milliliters of ascending methanol concentrations. After the last incubation, incubate the embryos at room temperature in 1.5 milliliters of 1%hydrogen peroxide and methanol for 30 minutes.

Then, wash the embryos for 10 minutes per wash in 1.5 milliliters descending methanol solutions followed by two five-minute washes in 1.5 milliliters of PBS. Next, wash the embryos in 1.5 milliliters of double distilled water for five minutes followed by a seven-minute wash in 1.5 milliliters of minus 20 degrees Celsius acetone. Wash the embryos in another 1.5 milliliters of double distilled water for five minutes followed by a five-minute wash in 1.5 milliliters of PBST with 1%DMSO or PBDT.

Incubate the embryos at room temperature in 1.5 milliliters of PBDT with 10%FBS on a rocker for two hours. Then, replace the PBDT and FBS with 1.5 milliliters of the primary antibodies for an overnight incubation at four degrees Celsius. The next morning, wash the embryos in 1.5 milliliters of PBDT, FBS and 0.1 molar sodium chloride for one minute with rocking followed by five 30-minute rocking washes in 1.5 milliliters of PBDT, FBS and sodium chloride.

After the last wash, wash the embryos one time in 1.5 milliliters of PBDT and FBS only for 30 minutes with rocking followed by an overnight incubation in 200 microliters of the appropriate secondary antibodies at four degrees Celsius protected from light. The next morning, quickly wash the embryos two times in 1.5 milliliters of PBDT followed by a 15-minute incubation in 1.5 milliliters of DAPI on a rocker. Then, wash the embryos three times in 1.5 milliliters of PBDT with FBS and sodium chloride for 15 to 20 minutes per wash and store the embryos in 1.5 milliliters of PBDT at four degrees Celsius protected from the ambient light.

To image the zebrafish kidneys, lay the embryos lateral on glass slides in approximately 10 microliters of PBDT. Using a pair of fine forceps and a dissecting microscope, squeeze the first embryo behind the eyes to remove the head and some of the yolk ball. Then, gently scrape away the remaining yolk ball from the embryo body.

While removing the yolk ball, be careful not to touch the yolk sac extension as the tissue of interest tears easily and is located directly above the extension. Use a thin tissue to remove the dissociated yolk and extra liquid from the slide. Then, add a drop of mounting medium to the remaining tail and position the tail laterally.

Flatten the tail with a cover slip and place the slide in a covered slide box. Then, use the 60X objective on a confocal microscope to image the kidney's cilia on the appropriate fluorescent channels. In the following representative images of a zebrafish embryo stained as just demonstrated, multiciliated cells can be identified based on visualization of the antisense riboprobe used to recognize ODF3B transcript expression, the gamete tubulin labeled basal bodies and the acetylated alpha tubulin labeled cilia.

Indeed, at this magnification of the embryonic pronephros, a multiciliated cell with ODF3B transcripts, multiple basal bodies and cilia can be observed. The adjacent monociliated cell can be distinguished by its single basal body and single cilium phenotype. While attempting this procedure, it's important to remember to use freshly fixed embryos and to keep the specimens protected from ambient light after the first antibody has been added.