Immunohistochemical Assay for Opsin Localization

Immunohistochemistry procedures were performed with polyclonal antibodies raised in rabbits against the last 42 amino acids of the C-terminal of human blue opsin (Sigma-Aldrich; AB5407), against the last 38 amino acids of the C-terminal of human red/green opsins (Sigma-Aldrich; AB5405), or raised in goats against a synthetic peptide with 20 amino acids of human blue opsin (Santa Cruz Biotechnology; sc-14363) (Table 1). The specificity of the antibodies for snakes was described previously (Hauzman et al., 2014 (link), 2017 (link); Bittencourt et al., 2019 (link); Gower et al., 2019 (link)). Double labeling with the antibodies against SWS1 and against LWS opsins showed differential labeling of distinct photoreceptor populations (Figure 2), further indicating the specificity of both antibodies for particular types of cones. We also assessed the specificity of the two anti-SWS1 antibodies, by incubating 12 μm retinal sections of B. jararaca and C. durissus obtained at −25°C with a cryostat (Leica, CM1100; Nussloch, Germany), with a mixture of both antibodies, rabbit anti-SWS1 (AB5407; 1:200) and goat anti-SWS1 (sc14363; 1:200). Immunofluorescence visualization showed a small number of small single cones labeled by both antibodies (data not shown), indicating the specificity of both antibodies against SWS1 cones.