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.