Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Whole mount immunohistochemistry is a relatively quick and simple technique that allows for the spatial and temporal observation of proteins directly in a tissue or organism using labeled antibodies. The utility of this technique is that it allows for the staining of an intact tissue or an organism without having to first section it and confocal microscopy can be used to generate a three-dimensional representation of the protein expression pattern. Immunohistochemistry is widely used in biomedical research to understand the etiology and molecular mechanisms that underlie disease.

It is also a valuable diagnostic tool to identify tumors in pathological samples. To begin, prepare spawning tanks filled with system water and with a mesh or slotted liner in place. Place adult zebrafish mixed sex pairs in groups in the tanks overnight.

Use a 14-hour/10-hour light/dark cycle with lights coming on at 9 a.m. The next morning, with the lights on, change the spawning tank water with fresh system water to remove feces. Once eggs are laid, return the adults to home tanks.

Collect the eggs by drawing them up using a transfer pipette. Transfer 50 eggs to each Petri dish filled halfway with embryo medium. Remove any opaque and cloudy eggs that are dead or failed to divide.

Incubate the dish of eggs at 28.5 degrees Celsius until 23 hours post fertilization. If embryos are older than 24 hours post fertilization, with a pipette transfer the embryos to 200 micromolar PTU in embryo medium to prevent melanogenesis. Under a stereo microscope, dechorionate the unhatched embryos using ultra fine tip forceps.

Using fire-polished Pasteur pipettes to minimize damage, transfer them into glass or plastic Petri dishes coated with 1-2%agarose dissolved in embryo medium. Then transfer the embryos to 1.5 milliliter centrifuge tubes using a plastic or fire-polished pipette. Remove embryo medium with a micropipette.

Leave only enough liquid to just cover the embryos. Fix the embryos in 4%PFA for one to two hours with gentle rocking at room temperature. Next, wash the embryos three times in 1X PBS and 1%PBTriton for five minutes.

Use the embryos immediately or store at four degrees Celsius for up to one week. For long-term storage, dehydrate and store the embryos in 100%methanol at minus 20 degrees Celsius. To rehydrate the embryos, perform serial incubations five minutes each at room temperature with decreasing amounts of methanol and PBS and PBTriton for the final wash.

Proceed with the embryo preparation according to the manuscript. Select a blocking solution matching the secondary antibody host species, for example 10%goat serum in PBTriton with or without two milligrams per milliliter of BSA. Add 0.5 milliliters of the blocking solution in the tube containing the embryo and place the tube on a rocker for one to three hours at room temperature.

Then incubate the embryo in primary antibody diluted in blocking solution and PBTriton overnight at four degrees Celsius while rocking. In the morning, wash the embryo five times in PBTriton for 10 minutes each at room temperature while rocking. Then select a secondary antibody based on the host species of the primary antibody and the desired wavelength at 488 nanometers.

Add the secondary antibody diluted in blocking solution into the tube containing the embryo. Cover the tube with aluminum foil and incubate for two hours at room temperature while rocking. After that, wash the embryos three times in PBTriton for 10 minutes each at room temperature while covered with aluminum foil and rocking.

With a pipette, transfer the embryos to a 50%glycerol solution in PBS over a bed of 2%agarose in embryo medium. To remove the yolk, transfer 200 microliters of 1X PBS to a depression slide or a plain glass slide. Use a plastic transfer pipette to move one or more embryos to the PBS droplet.

Use ultra fine forceps and double zero insect pins to break apart the yolk and very gently scrape yolk granules from the ventral surface of the embryo. Remove the yolk granules and replenish with PBS as needed. After mounting, place the sample on the microscope stage.

Locate the region of interest by selecting a relatively bright example. Adjust the camera exposure and gain so that the signal is sufficiently bright without saturating. When comparing between the experimental antibody-labeled embryos and control antibody embryos, use the same exposure settings.

This protocol of whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression using zebrafish development. The GluN1 subunit of the NMDA type glutamate receptor revealed expression of glutamate receptor subunits throughout developing zebrafish muscles at 23 hours post fertilization. Frozen sections of 72-hour post fertilization embryos were mounted on slides and immunostained for phospho-H3, a marker of proliferating cells.

Several cells expressed phospho-H3 and the expression was most notable at the ventricular zones. Mouse IgG control antibody and excluding primary antibodies revealed a low level of non-specific expression. It's important to select appropriate blocking solutions and secondary antibodies based on the primary antibodies host species to ensure detection and to minimize non-specific staining.

Immunohistochemistry must be verified to ensure that the signal observed is actually the protein of interest. Followup experiments usually involve using genetically or environmentally modified organisms. Immunohistochemistry grants researchers the ability to observe proteins in situ, greatly accelerating our understanding of numerous systems in both basic and applied biology particularly during embryo development.

Methanol and formaldehyde are toxic and should be handled carefully. Wastes need to be collected and disposed of according to institutional procedures.