Permafluor permount mounting medium

Neurons attached to poly-lysine coated coverslips were rinsed once with PBS and fixed for 15 min on ice in 4% PFA made in PBS. After fixation, the neurons were washed with PBS twice and then incubated for 30 min in blocking solution (i.e. PBS containing 5% goat serum and 0.025% Triton X-100) followed by 2 hour incubation with primary antibody and 15 min with Alexa 546- or Alexa 633-conjugated secondary antibodies (Thermo Scientific) respectively, diluted in blocking solution. The coverslips were then mounted on glass slides with permafluor permount mounting medium (Thermo Scientific) and analyzed using a Zeiss upright confocal microscope (LSM7) with a 63x/1.32–0.6 oil-immersion objective. Images were collected using Zen software and processed using Adobe Photoshop software. All digital manipulations were equally applied to the entire image.

Experimental samples were obtained from mice post-mortem. No experiments were performed on live animals for this study. Animals were deeply anesthetized to avoid any pain and sacrificed mechanically either by decapitation or exsanguination. The protocol was approved by Virginia Tech IACUC committee (IACUC #14–148). All animal procedures were performed in accordance with the guidelines for the animal care of laboratory animals issued by Virginia Tech. Three months old mature adult mice were deeply anaesthetized (ketamine/xylazine) and mouse hearts were canulated and perfused first with PBS (exsanguination) followed by 4% paraformaldehyde (PFA). The perfused mice were decapitated and brains were dissected out and fixed in 4% PFA overnight. The brains were cryopreserved by incubation in 30% sucrose solution for 48 hours. For sectioning, the brains were embedded in cryotek and 20 μm thick cortical sections were generated using a cryostat (IEC). The cortical sections were immunostained as floating sections; first they were permeabilized in 0.025% Triton X-100 and then blocked with 5% goat serum. Sections were stained with primary antibodies for two hours followed by secondary antibody incubation for 30 minutes. Finally, sections were mounted on slides using permafluor permount mounting medium (Thermo Scientific) and coverslips were sealed using nail polish.