Vectashield anti fade mounting media h 1000

After drug treatment, cells were cytospun (800 rpm, 8 min) to glass slides and were fixed for 15 min with 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4). After incubation for 20 min in 70% ethanol, the cells were stained with 20 μg/mL propidium iodide with 100 μg/ mL RNase A for 15 min in the dark. Finally, slides were mounted with Vectashield anti-fade mounting media (H-1000, Vector Laboratories, Inc., Burlingame, CA, USA). Images were taken using a Zeiss 780 confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA).

After the recordings, the brains were dissected out and fixed in 4% PFA at room temperature for 30-60 mins. Quick washes with PBS were followed by incubation in PBS containing 0.2% Triton-X (PBS-T) for 1 h. Then, the brains were incubated in 5% normal goat serum at room temperature for 2–3 h or kept at 4 °C for overnight incubation. After this, the samples were incubated in 1:30 rat anti-DN-cadherin (DN-EX #8, Developmental Studies Hybridoma Bank) and 1:500 rabbit anti-GABA (A2052, Sigma-Aldrich) or 1:200 rabbit anti-Lucifer yellow (A5750, Molecular Probes) for 1–2 days at 4 °C. Brains were washed in PBS-T for several hours at room temperature and incubated with 1:500 goat anti-rat with Alexa 405 (ab175671, Abcam), 1:500 goat anti-rabbit with Alexa 633 (A21070, Molecular Probes) or goat anti-rabbit with Alexa 488 (A11008, Molecular Probes) and 1:10⁵ Streptavidin with Alexa 488 (S11223, Molecular Probes) or Streptavidin with Alexa 568 (S11226, Molecular Probes) for 1–2 days at 4 °C. Then the brains were washed multiple times with PBS and PBS-T for 3–4 h and mounted in Vectashield anti-fade mounting media (H-1000, Vector Laboratories). Morphological images were obtained using Nikon A1R MP+ microscope and NIS-C software version 5.1 (Nikon).