Basescope v2 assay

The in situ hybridization (ISH) BaseScope^™ v2 assay (Advanced Cell Diagnostics (ACD)) was performed on 5 μm-thick formalin-fixed paraffin-embedded sections of adult human eyes according to the BaseScope^™ detection reagent kit v2 ACD protocol. Probes against the ubiquitously expressed isomerase PPIB were used as positive control, and probes against bacterial DapB were used as negative control. Six custom probes of 18–25 bp oligonucleotide sequences were designed by ACD for highly specific and sensitive detection of human CIDEC RNA. After deparaffinization in xylene and endogenous peroxidase activity inhibition by H₂O₂ (10 min), sections were permeabilized and submitted to heat (15 min at 100°C) and protease IV treatment (20 min at 40°C). After probe hybridization for 2 hours at 40°C, the signal was chemically amplified using the kit reagents and detected using the FastRED dye. The sections were then counterstained with Hematoxylin and mounted using VectaMount (Vector Labs, H-5000).