Brain Distribution of Pre-miR-7-2 by FISH

To evaluate the cellular distribution of pre-miR-7-2 in the brain, FISH assay was performed based as our previous description with some modifications [23 (link)]. Briefly, before hybridization incubation, all solutions were prepared with diethylpyrocarbonate-treated water. After deparaffinization and rehydration, tissue sections were treated by pepsin digestion. Sections were next incubated or heated in the microwave, and then were incubated with hybridization cocktail containing miR-7-2 probe (1:1000; EXIQON; no. 38485–01) at 42 °C for overnight. Next, the sections were washed in PBS and incubated with a secondary antibody of Cy3 conjugated goat-anti-rabbit IgG (1:250; Invitrogen) in the dark, at room temperature for 1 h. Then, the slides were rinsed with PBS-T three times, for 5 min each and counterstained, mounted with Slow Fade Gold Antifade Reagent with DAPI (1:1000) in the dark, at room temperature for 10 min, before examination by fluorescence microscopy (Zeiss Axioplan 2).