Psd 9

Mice were anesthetized and transcardially perfused with filtered PBS (Sigma-Aldrich) and 4% PFA (Sigma-Aldrich)/PBS. Brains were left overnight in the same fixing solution and transferred to 30% sucrose in PBS for 72 hours. Coronal brain sections (40 µm) of perfused mice (Leica CM1950 Cryostat, Leica Biosystems) were stored at −20 °C in anti-freeze buffer [PBS, 20% sucrose (Sigma–Aldrich), 15% ethylene glycol (Sigma–Aldrich), 0.05% NaN3 (Sigma–Aldrich)]. After washing from the buffer and incubation with 5% NDS (Jackson ImmunoResearch, 017–000-121) (in PBS), the slices were incubated with primary antibody (cofilin, Cell Signaling, cat. 5175S, 1:400; PSD-9, Santa Cruz, sc-6926, synaptotagmin, Novus Biologicas, NB100–1938, 1:1000) in TBS with 0.3% Triton (TBST) and 5% NDS. The next day, the sections were washed with TBST and incubated with secondary antibodies (Invitrogen, Alexa Fluor 488 #A21206, Alexa Fluor 568 #A10037, Alexa Fluor 647 #A21447). Next, the slices were washed with PBS, mounted on microscopic slides and covered with DAPI containing medium (Fluoromount-G, Invitrogen, #00–4959-52). The fluorescent staining was photographed with a confocal microscope (Zeiss LSM800, magnification 63x) by the experimenter blind to the experimental groups. For each immunostaining all pictures were taken with the same settings.

Mice were anesthetized and transcardially perfused with filtered PBS (Sigma-Aldrich) and 4% PFA (Sigma-Aldrich)/PBS. Brains were left overnight in the same fixing solution and transferred to 30% sucrose in PBS for 72 hours. Coronal brain sections (40 μm) of perfused mice (Leica CM1950 Cryostat, Leica Biosystems) were stored at −20 °C in anti-freeze buffer [PBS, 20% sucrose (Sigma–Aldrich), 15% ethylene glycol (Sigma–Aldrich), 0.05% NaN3 (Sigma–Aldrich)]. After washing from the buffer and incubation with 5% NDS (Jackson ImmunoResearch, 017-000-121) (in PBS), the slices were incubated with primary antibody (cofilin, Cell Signaling, cat. 5175S, 1:400; PSD-9, Santa Cruz, sc-6926, synaptotagmin, Novus Biologicas, NB100-1938, 1:1000) in TBS with 0.3% Triton (TBST) and 5% NDS. The next day, the sections were washed with TBST and incubated with secondary antibodies (Invitrogen, Alexa Fluor 488 #A21206, Alexa Fluor 568 #A10037, Alexa Fluor 647 #A21447). Next, the slices were washed with PBS, mounted on microscopic slides and covered with DAPI containing medium (Fluoromount-G, Invitrogen, #00-4959-52). The fluorescent staining was photographed with a confocal microscope (Zeiss LSM800, magnification 63x) by the experimenter blind to the experimental groups. For each immunostaining all pictures were taken with the same settings.