Citrate buffer antigen retrieval was used for paraffin sections. We blocked all the sections in 10% heat inactivated donkey serum in PBS plus 3% Triton X-100 for an hour, and performed all antibody incubations in the same solution at room temperature for an hour in block or overnight at 4°C.
Whole mount RPE was dissected from unfixed eyes and then fixed in cold (−20°C) methanol for at least 30 minutes before immunofluorescence staining. Whole mount buffer (3% Triton X-100, 0.5% Tween-20, 1% BSA in PBS) was used to block, wash, and for all antibody incubations. Briefly, RPE was rehydrated in PBS, blocked for an hour, and primary antibodies incubated at room temperature overnight. Following washes, secondary antibodies were incubated for 2 hours, washed in whole mount buffer twice, and PBS twice before being mounted and imaged.
Tissue from at least five mice of each genotype from at least three different litters was used for all analysis. All tissues were mounted in Vectashield (Vector Laboratories, Peterborough, UK), imaged by confocal microscopy (Nikon A1R; Nikon Corporation), and maximum intensity projections of z-stacks were created using NisElements AR Version 4.0 software. All images are representative of at least three animals.
For immunofluorescence, the following antibodies were used; rabbit anti-Slc9a8 (1:200; AbD Serotec, Kidlington, UK), mouse anti-rhodopsin (1:500; Merck Millipore UK Ltd., Watford, UK), rat anti-F4/80 (1:500; AbD Serotec, UK), rabbit anti-ML- Opsin (1:500; Merck Millipore), goat anti-S-opsin (1:500; Insight Biotechnology, Middlesex, UK), mouse anti-β-catenin (1:200; Sigma-Aldrich, Dorset, UK), rabbit-anti-ZO-1 (1:100; Invitrogen, Renfrew, UK), rat anti-transferrin receptor (1:200; Lifespan Biosciences, Inc., Seattle, WA, USA), and mouse anti-Golgin 97 (1:200; Invitrogen). Alexa Fluor secondary antibodies (1:500; Invitrogen) were used.