Immunostaining Protocol for Satb2 and GFP

Following viral transduction, slices were fixed overnight at 4°C in 0.1 M phosphate-buffer containing 4% formaldehyde. Then, slices were rinsed with phosphate-buffer saline (PBS), permeabilized with PBS/gelatin 0.2%/Triton 0.25%, and incubated overnight at 4°C with rabbit anti-Satb2 (1:1000, ab34735, Abcam; Lee et al., 2010 (link)) and chicken anti-GFP (1:1000, GFP-1020, Aves Labs; Tricoire et al., 2010 (link)). After washing in PBS, the respective immunoreactions were visualized with the following secondary antibodies: goat-anti-rabbit AlexaFluor 555 (1:1000, A-21430, Thermo Fisher Scientific) and goat-anti chicken AlexaFluor 488 (1:1000, A-11039, Thermo Fisher Scientific) incubated 1 h at room temperature. Sections were mounted with fluoromount-G (Southern Biotech) on slides for visualization. Images of immunostained material were acquired using a Leica TCS SP5 AOBS inverted confocal microscope with a 40× objective (40× HCX P APO CS NA 1.25–0.75/Oil) and LAS AF software (Leica Microsystems). Cell counting was performed using Image Pro Analyzer 7.0.0.951 (MediaCybernetics).