Ab116590

Paraffin‐embedded sections (4‐μm thick) were deparaffinized, dehydrated and treated with 1 M sodium thiocyanate. The sections were then digested in a pepsin solution, fixed in 4% formaldehyde, dehydrated by sequential immersion in 70%, 85% and 100% ethanol and air‐dried. The sections were incubated with a digoxin (DIG)‐labeled probe (5′‐DIG‐CTCCTGCCAGACTCCAGATGTCCAAGGTGCTCC‐3′ for human H19, 5′‐DIG‐CTTTCCCGTTCTCCGGCCATCGAAGCT‐3′ for rat H19 and 5′‐DIG‐AACCACACAACCTACTACCTCA‐3′ for rat let‐7b) followed by incubation with a DyLight 594‐conjugated IgG fraction (Abcam; ab96873) coupled with a monoclonal mouse anti‐DIG antibody (Abcam; ab116590). The nuclei were counterstained with DAPI. The sections were scanned and analysed as described above.

FISH was performed to detect the subcellular location of NONRATT023402.2. Brain sections were digested in a pepsin solution, fixed in formaldehyde, and dehydrated by gradient ethanol solutions. The sections were then incubated with a digoxin (DIG)-labeled probe (5′-DIG-AGTAACGCTGAGTCTCGTGAGTCTGGTTCCAT-DIG-3′), followed by incubation with a DyLight 594-conjugated IgG fraction (ab96873; Abcam) coupled with a monoclonal mouse DIG antibody (ab116590; Abcam;). Nuclei were then counterstained with DAPI.