Detection of Orexin and Cannabinoid Receptor Co-localization in Diet-Induced Obese Zebrafish

In the zebrafish brain, neurons express orexin and cannabinoid receptors that regulate metabolism.

In diet-induced obese, or DIO, zebrafish, these receptors increasingly co-localize to form heteromers, altering downstream signaling and impacting metabolic regulation.

Take cryosections of the brain tissue from both control and DIO zebrafish. Wash the sections with buffer to remove the embedding medium.

Treat with a permeabilization-blocking buffer to permeabilize the cellular membranes and mask non-specific binding sites. Wash to remove excess buffer.

Incubate with a primary antibody cocktail targeting the receptors. Wash to remove unbound antibodies.

Incubate with different fluorophore-labeled secondary antibodies that bind to the primary antibodies. Wash to remove unbound antibodies.

Add a fluorescent DNA-binding dye to label the nucleus. Then, apply a mounting medium and seal the sections with a coverslip.

Using a confocal microscope, detect the antibody fluorescence. Increased co-localization of the antibodies in DIO zebrafish compared to controls indicates higher receptor heteromerization.

First, rinse the sections three times for 5 minutes each with 0.1 molar phosphate buffer. Add the mix of primary antibodies to lutetium PBT, and incubate the sections overnight in a humid box at room temperature. The day after, rinse the sections three times for 5 minutes each with 0.1 molar phosphate buffer. Incubate sections at room temperature for 2 hours in an appropriate mix of secondary antibodies diluted in PBT.

Rinse the sections three times for 5 minutes each with 0.1 molar phosphate buffer. Dissolve 1.5 microliters of DAPI in 3 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 4 degrees Celsius.