Automated In Situ Hybridization of Zebrafish Retina

In situ hybridization (ISH) was performed on paraffin-embedded zebrafish retina sections. Clrn1 transcripts were detected using an automated Leica Bond platform with heat-induced Leica ER2 antigen retrieval buffer solution and RNAscope 2.5 LS Protease III digestion (Advanced Cell Diagnostics, Hayward, CA, USA), as previously described (32 (link)). Briefly, after hybridization to 20ZZ probes targeting the region comprising nucleotides 2–911 of Dr-clrn1 NM_001002671.1, a six-step amplification process was performed, followed by chromogenic detection using Fast Red (Advanced Cell Diagnostics). Images were collected with a fully automated widefield DMi8 Leica fluorescence microscope.