In Situ hybridization.
Section in situ hybridization was performed as previously described [61] using 20-μm cryosections from OCT-embedded tissue or 4-μm paraffin sections. All in situ hybridizations were performed with the mutant and wild-type control sections on the same glass slide. Riboprobes labeled with digoxygenin-tagged UTP (Roche, Basel, Switzerland) were detected with NBT/BCIP (Sigma, St. Louis, Missouri, United States). The sources of the individual riboprobes used in this study are described in Tables S1 and S2. Dissociated cell in situ hybridization was performed as described previously [62] using the same S-opsin digoxygenin-labeled probe used for section in situ hybridization. All images were captured on a compound microscope (Eclipse e1000; Nikon, Tokyo, Japan) using a CCD camera (DXM1200F, Nikon). S-opsin positive cells were quantitated on dissociated cell in situ hybridization as previously described [62]. Twenty fields were quantitated in this manner at 200× magnification for both rd7 and wild-type retinas.