Ab96873

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.

We seeded 1 × 10⁴ cells on an 18-mm cover glass (GMA81-018, MATSUNAMI, Bellingham, WA, USA). After being treated with 2.5-μM Dac for 5 days, cells were fixed in 4% formaldehyde, washed and then permeabilized with 0.25% Triton X-100 in PBS for 10 minutes. Cells were subjected to staining with primary antibodies mouse anti-dsRNA mAb J2 (1:200, 10010200; SCICONS, Szirák, Hungary) for 2 h and rabbit anti-Tom20 pAb (1:100; sc11415, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h, secondary antibodies Alexa 488-conjugated donkey anti-mouse (1:500, ab96873, Abcam, Cambridge, UK) and Alexa 594-conjugated goat anti-rabbit (1:500, ab96883, Abcam, Cambridge, UK) for 1 h and DAPI (D1306, Thermo Fisher Scientific, Waltham, MA, USA). After washing twice with PBS, cover glass was mounted with Fluoromount-G^® (0100-01, SouthernBiotech, Birmingham, AL, USA). Super-resolution imaging was performed using a confocal microscope (LSM 980 with Airyscan 2, Zeiss, Oberkochen, Germany). The fluorescence signal intensity of dsRNA was captured under lower magnification (100×) of the Olympus FV10i confocal microscope. Quantification of the dsRNA signal was obtained in three fields containing at least 17 cells for each group from three independent experiments. The quantification of fluorescent signal representing dsRNA level was normalized to cell number.