Probing Melanopsin and Melatonin Receptors in Retinal Neurons

Melanopsin was probed with a polyclonal antibody raised in goats against a sequence between amino acids 410–460 of rat melanopsin (SC-26962, Santa Cruz Bio-technology, Santa Cruz, CA, USA; 1:50). It has been used previously to identify ipRGCs [36 (link),37 (link)]. To label MT₁ receptors, we used a rabbit polyclonal antibody directed against a peptide corresponding to a region of the third intracellular loop (residues 223–236: (C) RVKPDNKPKLKPQD) of mouse MT₁ receptor (AMR-031, Alomone laboratories, Jerusalem, Israel; 1:500). The MT₂ receptor antibody was raised in rabbits, targeting the third intracellular loop (residues 232–246: (C) RKAKATRKLRLRPSD) of the mouse MT₂ (AMR-032, Alomone; 1:50). In some of our experiments, a rabbit anti-MT₂ receptor antibody raised against N-terminal extracellular domain of human MT₂ receptor (SAB2900212, Sigma, St. Louis, MO, USA; 1:200) was used to probe MT₂ immunoreactivity. The secondary antibodies were Alexa Fluor 555-conjugated donkey anti-goat IgG (for melanopsin) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (for MT₁/MT₂) (Invitrogen, Carlsbad, CA, USA; 1:200). In the experiments demonstrating the cytoplasmic staining of melatonin receptors, Alexa Fluor 555-conjugated wheat germ agglutinin (WGA, Invitrogen; 10 μg/ml) was used to label the plasma membrane of retinal neurons.