The overall goal of this procedure is to detect differentiated interrenal steroidogenic cells in the developing zebrafish through a simple whole-mount enzymatic activity assay. This staining procedure can help answer key questions regarding the molecular and cellular mechanisms of interrenal gland formation and malformation. The main advantage of this staining assay is that it's rapid, simple, and reliable, making it a powerful tool for genetic or chemical screens in zebrafish.
This procedure can be performed on trangenic fluorescent reporter lines, marking the kidney and the blood vessels so that multiple tissues in the same embryo can be analyzed. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, the vascular microenvironment of the interrenal tissue can also be visualized. After preparing stock solutions for three beta-Hsd activity staining according to the text protocol, place developing zebrafish embryos in 0.03%PTU in egg water to inhibit pigment formation.
Fix dechorionated embryos, or larvae, in 2%or 4%PFA in PBS with 0.1%Tween 20, or PFAT. With PBST, wash the embryos four times for 10 minutes each, ensuring that the samples do not dry out between washes. Prior to the staining reactions, freshly prepare parts A and B in separate tubes, then vortex and centrifuge the solutions.
It is essential to prepare parts A and B in separate tubes. Adding all the components straight into one single tube will cause severe precipitation right away. Add the entire tube of part A to the entire tube of part B.Mix well, and immediately distribute one milliliter into each microfuge tube containing fixed and washed embryos.
Use aluminum foil to wrap the tubes, and place them at room temperature on a rotator or a rocking platform with gentle shaking. Empirically determine the reaction time by using a microscope to monitor chromogenic signals. Slight precipitations will develop over time that will not interfere with the reaction process.
Stop the reaction by using PBST to wash the embryos four times for 10 minutes each, then post-fix the stained embryos with 4%PFAT at room temperature for 60 minutes. For whole-mount microscopy, use 50%glycerol in PBS to clear the embryos. Then, after orienting them with the dorsal side facing up, observe the samples using an upright microscope under bright field illumination.
De-yolk the embryo, and use it to prepare a flat mount. Then, capture a high-resolution image of the interrenal tissue morphology. To analyze the vascular microenvironment of the steroidogenic tissue, post-fix the stained embryos with 2%PFA.
Supplement it with 1%Triton X-100 at four degrees Celsius for one hour, and use PBST-X to wash the embryos at room temperature four times for 10 minutes each. To embed the embryos, prepare 4%low-melting agarose by dissolving agarose in PBS at 95 degrees Celsius. Then, aliquot in microcentrifuge tubes, and store at four to eight degrees Celsius.
Melt an aliquot of 4%low-melting agarose at 70 degrees Celsius for 10 minutes, then keep the aliquot at 47 degrees Celsius. Using a detached cap from a 1.5%milliliter microcentrifuge tube as a mold, embed an embryo in molten agarose, and allow it to cool until solid. To carry out vibratome sectioning, adhere a piece of paper tape to the dry platform of the vibratome.
Then, use a razor blade to remove the hardened agarose block from the mold. Apply superglue to the paper tape on the platform, and fix the agarose block onto the paper tape with the anterior side of the sample facing up. Next, trim the agarose block to a trapezoidal prism shape with approximately four to eight millimeters on each side of the upper and lower facets respectively.
Use a dropper filled with cold PBS with 0.5%Trident X-100 to rinse the agarose block. Then, use a vibratome according to the manufacturer's instructions to prepare 100 micrometer sections. With a 26-gauge needle, remove cut sections from the vibratome and transfer them to a spot plate containing 500 microliters of cold 0.5%PBST-X per well.
Collect the three beta-Hsd activity positive tissue sections by checking under a dissection microscope, and use a pipette tip to transfer the sections into a 96-well plate containing cold 0.5%PBST-X. Further stain and image the sections according to the text protocol. To determine how steroidogenic interrenal tissue codevelops with the pronephric kidney glomerulus and its nascent vasculature, the three beta-Hsd enzymatic activity assay was performed on a double transgenic zebrafish embryo, expressing both kidney-specific and endothelial-specific fluorescence.
As shown here, three beta-Hsd positive steroidogenic tissue is located right of the midline and immediately coddle to the pronephric kidney glomerulus. Some steroidogenic cells also start migrating across the midline. These vibratome sections from transgenic embryos expressing endothelial GFP subjected to the three beta-Hsd assay were analyzed by immunohistochemistry to detect fibronectin and its downstream effector, phosphorylated focal adhesion kinase.
The peri-dorsal aorta deposition of fibronectin is essential for supporting interrenal vessel growth. For the fish embryo or larva, the whole-mount histochemical staining can be done within one day if it is performed properly. Although this staining method is simple and rapid, it is important to remember that it is detecting only one single enzyme of the steroidogenic pathway.
If aiming for a developmental analysis of the interrenal tissue, in addition to this assay, in situ habitization will have to be performed to detect the expression of other critical genes involved in steroidogenesis.
