The purpose of this project was to see if melatonin receptors are present on the photoreceptors of Fiddler Crabs. Melatonin is a homone that is expressed primarily at night. It is involved in regulating circadian rhythms and other physiological processes. The fiddler crab is an excellent model to use because the hormones that modulate the neurons in their visual system are similar to those found in humans.
The photoreceptors were extracted from the cornea of the eyestalk, placed in glass bottom dishes, and fixed in 3.7% formaldeyde for 15 minutes. The cells were washed with 1 X PBS (phosphate buffer saline) and treated with 1 M of glycine for 15 minutes. The photoreceptors were immediately washed with 1 X PBS and treated with 0.5% triton-X 100 for 10 minutes. The cells were washed again with 1 X PBS and treated with 1% serum for 30 minutes. NOTE: All procedures were carried out at room temperature. The serum should be the same species as the secondary antibody. In this
project, I used Donkey!
The cells were rinsed with 1 X PBS and incubated for 60 minutes with 200 microliters of MEL 1A-R antibody (goat: 1:50 dilution) in the dark. After rinsing with 1 X PBS, the cells were treated with the secondary antibody (Donkey Anti-goat; 1:100 dilution) for 30 minutes and washed with 1 X PBS.
Here are the results using tissue from whole eyestalks.
Drum roll PLEASE! Check out the video!:
Therefore, early microscopy studies suggest that melatonin receptors may be present on the photoreceptors. Future experiments will involve western blotting and sequencing to confirm their presence.