Alexa 488 conjugated secondary fluorescent antibody

After behavioral tests, mice were then deeply anesthetized with pentobarbital (100 mg/kg, intraperitoneal) and transcardially perfused with saline followed by 4% paraformaldehyde in a 0.1 mol/L phosphate buffer (Sigma‐Aldrich). The brains were removed and post‐fixed in the 4% phosphate‐buffered paraformaldehyde at 4°C overnight, and were then cryo‐protected with 30% (w/v) sucrose. Coronal 30‐μm slices of brains were cut on a freezing sliding microtome (Leica). Brain slices were processed for immunofluorescence for PV (1:1000, Swant PV27), then incubated with primary antibody diluted in phosphate‐buffered saline with 0.15% Triton X‐100 overnight at 4°C. Then they were rinsed with phosphate‐buffered saline and incubated with an Alexa‐488 conjugated secondary fluorescent antibody (1:400, Jackson Immuno Research) at 1 μg/mL for 2 hours at room temperature. The slices were mounted on slides by the Vectashield Mounting Media (Vector Labs) after rinsing, and the immunofluorescence was accessed by using an Olympus microscope (BX61).

Mice were killed and transcardially perfused with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS). The brain was then removed, fixed in 4% paraformaldehyde overnight, and then equilibrated in 30% sucrose at 4 °C. Coronal sections (30 μm) were cut on a freezing microtome (Leica CM3050, Germany). After rinsing with 0.5 % Triton X-100 (vol/vol) in 0.1 M PBS for 30 min and blocking with 10% (wt/vol) normal bovine serum for 2 h at room temperature, sections were incubated with the following primary antibodies diluted in PBS with 0.15% Triton X-100 overnight at 4 °C: anti-GAD65/67 (Millipore AB1511), anti-nNOS (ThermoFisher 61-7000), and anti-PV (Swant PV27). After rinsing with PBS, sections were incubated with Alexa-594 or Alexa-488-conjugated secondary fluorescent antibody (Jackson ImmunoResearch, 711-585-152 or 711-545-152) for 2 h at room temperature. After rinsing, the sections were mounted on slides using Vectashield Mounting Media (Vector Labs) and confocal images were captured (Olympus FV-1000). Fluorescent expression of optogenetic or chemogenetic virus was examined for all mice. If the optical fiber was located above (within 0.5 mm) the targeted region with the correct viral expression, the corresponding mouse was taken into statistical analysis.