RNA in situ hybridization

RNA in situ hybridization assays were performed on cryo-sections. Antisense probes and sense (control) probes for the tested transcripts were generated by polymerase chain reaction (PCR) on human genomic DNA using primers that were tailed with sequences recognized by the RNA polymerases T3 and T7 (Supplementary Table S3). PCR products were purified and used as templates for in vitro cRNA transcription. To detect the probe, a hapten tag (digoxigenin-labeled Uridine-5′-triphosphate (UTP)) was incorporated into the RNA during the in vitro transcription reaction (DIG RNA labelling kit; Roche). RNA ISH experiments on human eye sections using cRNA probes were performed as previously described (23). Hybridization was performed at 65°C.