Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Currently, immunohistochemistry is used with increasing frequency to identify the presence of specific molecular markers and their changes in case of pathologies, giving root to the possibility to perform quantitative analysis. Immunohistochemistry, mainly the immunofluorescence technique, is used in the larval and the adult zebrafish animal model, but little is known about a good standard immunohistochemistry protocol for zebrafish. Here, we describe and illustrate a protocol that can be used to study the evolutionary conservation of important proteins in adult zebrafish.

A good protocol for immunohistochemistry in adult zebrafish can help to underlie the usefulness of this animal model to study the morphological expression and distribution of highly conserved biomolecules. This method can also help to analyze possible alterations of these biomolecules due to pathological conditions correlated to human physiopathology. Demonstrating the procedure will be Dr.Lea Tunisi, a PhD student from my laboratory.

To begin this procedure, transfer the prepared frozen tissue blocks to a cryostat at minus 20 degrees Celsius. Cut the block in coronal or sagittal sections of 10 micrometers. Then, collect the tissues in alternate serial sections onto adhesive glass slides suitable for immunohistochemistry, and store them at minus 20 degrees Celsius until ready to proceed.

First, use a solvent-resistant pen to demarcate the tissue area on the slide. Rinse the sections three times for five minutes each using 0.1-molar phosphate buffer. Next, dissolve 0.3 milliliters of Triton X-100 in 100 milliliters of 0.1-molar phosphate buffer to prepare Triton X-100 0.3%Dissolve 0.1%normal donkey serum in phosphate buffer containing 0.3%Triton X-100 to prepare the blocking solution.

Incubate the sections with the solution of normal donkey serum dissolved in permeabilization buffer PB-Triton X-100 0.3%for 30 minutes at room temperature to permeabilize the cell membrane and block the nonspecific binding sites. First, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer. Add the mix of primary antibodies diluted in PB-T, and incubate the sections overnight in a humid box at room temperature.

The day after, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer. Incubate sections at room temperature for two hours in an appropriate mix of secondary antibodies diluted in PB-T. To begin, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer.

Dissolve 1.5 microliters of DAPI in three milliliters of phosphate buffer to prepare the nuclear dye DAPI. Counterstain the sections in the dye. Then, use mounting medium to coverslip the slides to stabilize the tissue and stain for long-term usage.

Fluorescent samples can be stored in the dark at four degrees Celsius. Repeat the immunofluorescence protocol, and either omit the primary or secondary antibody, or substitute the primary or secondary antiserum with phosphate buffer to prepare the negative control. Next, repeat the immunofluorescence protocol, and pre-absorb each primary antibody with an excess of the relative peptide.

Repeat the immunofluorescence protocol on slices of mouse brain to prepare the positive control. Using a confocal microscope equipped with an X-Y-Z motorized stage, a digital camera, and acquisition and analysis software, observe and analyze the immunostained sections. Acquire digital images with the 5x, 20x, and 40x objectives.

Take images of each section at low magnification in each of the available channels to compose a low-magnification montage. Normalize the fluorescence images to maximum contrast and overlay. Throughout the area of interest, collect serial Z-stacks of images through six focal planes with focal steps of one to 1.8 micrometers.

Perform this collection for each channel separately. After this, use imaging deconvolution software to deconvolve images by application of 10 iterations, and collapse the serial Z-plane images into a single maximum projection image. Use an appropriate image editing software to adjust the micrographs for light and contrast.

Immunohistochemical analysis of OX-A and OX-2R distribution in the gut of adult zebrafish shows different localization sites of OX-A and OX-2R and their increases in expression in the intestinal cells of DIO zebrafish. An intense brown staining for OX-A is observed in the cells of the medial and anterior intestine. The immunoexpression of OX-A gives clear signals in the different gut compartments, decreasing from the anterior toward the medial intestine.

Similar results are observed for OX-2R immunoexpression in the intestine of DIO and control diet zebrafish. An increased OX-A signal in DIO adult zebrafish is accompanied by the overexpression of OX-2R in other intestinal compartments. Accurate analysis of immunofluorescent images shows the increase of OX-2R/CB1R colocalization in the gut of DIO adult zebrafish compared to the control diet zebrafish.

A similar situation is observed in different brain regions, such as the dorsal telencephalon, hypothalamus, optic tectum, torus lateralis, and diffuse nucleus of the inferior lobe. By double immunostaining with orexin-A and CB1R colocalization, it is observed that there was an increase of colocalization in the orexinergic neurons of the hypothalamus, accompanied by an increase of orexin-A fluorescent signal. These results show how double immunofluorescence can help to identify physiologically conserved protein expression, colocalization of target proteins, and their distribution and/or expression changes in different pathological conditions.

Using immunofluorescence, we can better understand the codistribution and coexpression of biomolecules, their possible interactions, and we can also have a visible image of their changes in case of different pathologies. Moreover, the neuroanatomical distribution of metabolic enzyme expression or receptors may further reveal the role of protein signaling within tissues. The present technical work introduced the immunofluorescence approach to study two highly conserved system, orexin and endocannabinoids, in using an adult zebrafish model.

Using this protocol described here for the first time in the brain of adult zebrafish, the anatomical distribution and coexpression of orexin-2 receptors and the endocannabinoid-CB1 receptors hasn't been determined. We recommend the protocols described here for immunohistochemistry experiments and to detect spatial distribution and organization of receptors in peptides in adult zebrafish to better understand their functions.