Am9763

Neurons were plated on PDL-coated 28-mm glass bottom dishes (Invitrosci) and fixed 7 days later with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized with cold 70% ethanol overnight at 4°C and washed the following morning for 5 minutes with wash buffer (25% formamide, 2X SSC). Neurons were stained with 40 20-nucleotide-long fluorescently labeled (Alexa-647) oligonucleotides designed against the Aag transcript overnight at 37°C in a heavily humidified chamber in hybridization buffer (100 mg/mL dextran sulfate, Sigma, D8906: 0.5 mg/mL E.coli tRNA, Sigma, R4251: 0.5 mg/mL ssDNA, Sigma D9156: 1 mg/mL Ultapure BSA, Ambion, AM2616: 10 mM VRC, New England Biolabs, S1402S: 25% formamide, Ambion, AM9342: 2X SSC, Ambion, AM9763). Finally, cells were stained with DAPI (2 μg/mL) in wash buffer for 30 minutes at 37°C before imaging. All the previous steps were done in nuclease-free solutions to avoid RNA degradation. We occasionally see faint transcripts in Aag^-/- cells due to initiation of endogenous transcription before hitting the inserted cassette that disrupts the gene.
Images were taken with a Hamamatsu ORCA-R² CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].

Freshly cut 3 μM FFPE samples were baked at 70 °C for 15 min. After deparaffin and rehydration, samples were treated with boiling antigen-unmasking solution for 1 h. Samples were cooled down to room temperature, and then treated with 0.2 × SSC buffer (Ambion; AM9763) with gentle shaking at room temperature for 20 min. Primary antibody incubation was done with monoclonal antibody S9.6 (Karafast; ENH001) at 1:100 dilution in PBS containing 1% normal goat serum and 0.5% Tween-20 at 37 °C overnight. After primary antibody incubation, samples were washed three times with PBS containing 0.5% Tween-20, and then incubated with Alexa-488-conjugated secondary antibody at 1:1,000 dilution in PBS containing 1% normal goat serum and 0.5% Tween-20 at 37 °C for 2 h. Samples were washed two times with PBS containing 0.5% Tween-20, two times with PBS, and then mounted with Vectashield mounting medium with DAPI. For samples pretreated with RNase H, an overnight treatment of RNase H (NEB; M0297S) was carried out after 0.2 × SSC treatment. Samples were washed three times with PBS before primary antibody incubation.