Planapo 63x 1.4 na

Cells were washed with phosphate-buffered saline (PBS; Oxoid, BR0014G), then fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 (Sigma Aldrich, T8787) solution or 100 μg/ml digitonin solution (Life Technologies, BN2006) for 20 min, as indicated. Cells were incubated with the appropriate primary antibodies for 1 h, washed 3 times with PBS, and then incubated for 30 min with appropriate Alexa Fluor 488-conjugated (Life Technologies, A21202), Alexa Fluor 555-conjugated (Life Technologies, A31272) or Alexa Fluor 647-conjugated (Life Technologies, A21245) secondary antibodies and then washed again 3 times in PBS. Nuclei were stained with a solution of 1.5 μM of 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, D9542) in PBS for 5 min. Coverslips were mounted in Fluorescence Mounting Medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica Microsystems, Germany, Mannheim) installed on an inverted LEICA DMI 6000CS microscope (Leica Microsystems, Germany, Mannheim) and equipped with an oil immersion PlanApo 63X 1.4 NA objective. Images were acquired using the LAS AF acquisition software (Leica Microsystems).

Confocal images at different treatments and time points were acquired with a PLANAPO 63x/1.4 NA oil immersion objective by using a TCS SP8 AOBS laser‐scanning confocal microscope (Leica Microsystem) equipped with an Ar/Ar‐Kr 488, 561, and 633 nm diode laser lines and Hybrid detectors. The parameters for acquisition (561 laser line power and Hybrid detector gain) were defined on control conditions and kept constant for all the samples. For the acquisition, we focused on areas where DiI diffused and dendritic spines were clearly visible. Every image included dendritic segments (from 2 to 7) characterized by medium and small thickness, equally represented in the analysis, suggestive of proximal and distal ramifications. The 3D reconstruction of DiI‐filled dendrites was performed using the Simple Neurite Tracer, a dedicated module of Fiji software, and the total dendrite length was measured. The number of dendritic spines was evaluated using the ImageJ program (http://imagej.nih.gov/ij/) and the spine density was then calculated. Mean spine density changes at 48/72 h after the two different treatments were calculated (control values set to 1).