Immunohistochemical (IHC) analysis was performed on 5 µm paraffin embedded sections using standardized lab protocols [19 (link)]. Sections were deparaffinated in a series of xylene and ethanol dips followed by a rinse in PBS. Antigen sites were unmasked by soaking the sections in sodium citrate buffer. Sections were blocked with 2% normal horse serum (S-2000 Vector Labs, CA) in 1× PBS at room temperature for an hour. The primary antibodies rhodopsin (mouse monoclonal, Millipore MAB5356), blue opsin (rabbit polyclonal, Millipore AB5407), and green/red opsin (rabbit polyclonal, Millipore AB5405) were applied at 1:200 dilutions in PBS and incubated at 4 °C overnight. The following day sections were rinsed with PBS and the corresponding secondary antibody was applied in the dark in a 1:400 dilution in PBS: Alexa Fluor 488 goat anti-mouse (Invitrogen A11001) for rhodopsin and Alexa Fluor 488 goat anti-rabbit (Invitrogen A11008) for green and blue opsin slides. These slides were then incubated for an hour in the dark and finally nuclei were labeled with 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, dilactate, Invitrogen D3571). Opsin labeling was visualized and imaged using a Leica DMI6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany) [19 (link)].
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