Quorum spinning disk confocal microscope

Approximately 3 weeks following surgery mice were transcardially perfused with Phosphate-Buffered Saline (PBS, pH 7.4), followed by 4% paraformaldehyde in phosphate buffer. Brains were extracted and postfixed overnight at 4°C and subsequently cryoprotected with PBS containing 30% sucrose. Coronal 40 μm thick sections were obtained using a cryostat (Leica, Germany). The sections were slide-mounted, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, and coverslipped with Aquamount (Polysciences Inc., Warrington, PA, USA). The wide-field fluorescent images were captured with a 4× objective on a fluorescent microscope (Olympus, Japan). eYFP and red fluorescent retrobeads signals were captured using an U-MWIBA3 filter cube (Ex460- 495, Em510-550, DM505) and an U-MWIG3 filter cube (Ex530-550, Em575IF, DM570), respectively. Confocal images were captured through a Quorum spinning disk confocal microscope (Zeiss) using a 20× objective lens and were subsequently analyzed with Volocity Software (Perkin Elmer). eYFP and red fluorescent retrobeads signals were excited with 491 and 561 nm laser, respectively. Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA) was used to adjust the brightness and contrast of representative sections.

After behavioural testing, mice were transcardially perfused with phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in phosphate buffer. Brain tissue was extracted and postfixed overnight at 4 °C. The brains were then cryoprotected using a 30% sucrose in PBS solution. Coronal 40 µm-thick sections were collected using a cryostat (Leica, Germany). The sections were slide-mounted, counterstained with 4′,6-diamidino-2-phenylindole 135 for 5 min and subsequently coverslipped with Aquamount (Polysciences, Inc., Warrington, PA). Wide-field fluorescent images were captured using a 4 × objective lens on a fluorescent microscope (Olympus, Japan). Confocal images were captured using a × 20 and × 60 objective through a Quorum spinning disk confocal microscope (Zeiss, Germany). Adobe Photoshop CS6 (Adobe Systems, Incorporated, San Jose, CA) was used to adjust the brightness and contrast of representative sections.

Mice were transcardially perfused with 0.1% phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde (PFA). The brain was then extracted and post-fixed in 4% PFA overnight. Next, the tissue was stored in a 30% sucrose solution at 4 °C for 72 h. Coronal sections (40 µm) were obtained using a cryostat (Leica, Germany), slide-mounted and counterstained with DAPI (4′,6-diamidino-2-phenylindole) for 5 min before adding a coverslip with Aquamount (Polysciences, Inc., Warrington, PA). Wide-field fluorescent images were captured using a ×4 objective lens on a fluorescent microscope (Olympus, Japan). Confocal images were captured using a ×20 and ×60 objective through a Quorum spinning disk confocal microscope (Zeiss, Germany). Adobe Photoshop CS6 (Adobe Systems, Incorporated, San Jose, CA) was used to adjust the brightness and contrast of representative sections.