Sp8 laser point scanning confocal microscope

Postnatal animals were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA), their brains were dissected out and post-fixed in 4% PFA overnight at 4°C. Brains were sliced into 50 μm sections on a vibratome (Leica Microsystems VT1200S). Sections were prepared and placed in blocking solution (0.3% Triton (Amresco), and 10% goat serum) for 1-2 h at room temperature. After washing with PBS, sections were incubated sequentially with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature (RT). DAPI (4’,6-diamidino-phenylindole, Invitrogen) was added to the secondary antibodies solution. The sections were mounted using ProLong Gold Antifade Mountant (Invitrogen). Cryosections: Brains were cryoprotected in 30% sucrose/PBS overnight at 4°C after post-fixation. Brains were embedded in O.C.T. compound (Sakura), frozen on dry ice and stored at −80°C. Samples were sectioned at 20 μm on a cryostat (ThermoFisher CryoStart NX70). Images were acquired using a Leica SP8 laser point scanning confocal microscope, 10X, 20X, 40X, 63X and 100X objectives were used and images were further analyzed using Fiji, brightness and contrast were adjusted as necessary for visualization, but the source images were kept unmodified.