Fluorescence in situ hybridization procedures (Panomics) used to amplified low copy mRNA targets were used to visualize Arc and 18S mRNA within neurons (Taylor et al., 2009). PDMS top pieces were removed and cells attached to the glass coverslips were fixed immediately using 4% formaldehyde/4% sucrose in PBS for 20 min, followed by ethanol dehydration. After rehydration, cells were permeabilized using a detergent solution (Panomics) for 5 min. We used proteinase K (1:6000; Panomics) to digest proteins for 10 min at room temperature, followed by in situ hybridization using Arc (anti-sense or sense) and 18S probes designed by Panomics, following the manufacturer's instructions. Briefly, probes were diluted 1:100 in hybridization buffer supplied by Panomics, incubated at 40° C (3-5 h), washed, hybridized with pre-amplification oligos (1:100) at 40° C (25 min), washed, hybridized with amplification oligos (1:100) at 40° C (15 min), washed, and finally hybridized with the label oligos (1:100) at 40° C (15 min).
