Ab185921

Mice were euthanized using pentobarbital (120 mg/kg, i.p) followed by transcardiac perfusion with 0.1 M phosphate buffer followed by 4% p-formaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Brains were removed, post-fixed in 4% PFA overnight and placed in 30% sucrose solution for 48 h. Coronal series sections (20 μm) were sliced on a freezing microtome (Leica SM2000R, Leica Microsystems GmbH, Buffalo Grove, IL, USA) and stored in a cryoprotective solution. Double-label immunofluorescence was performed on free-floating sections and incubated overnight at 4°C with the following primary antibodies: Goat anti-chicken GFP (ab13970, Abcam, Cambridge, United Kingdom) and Goat anti-rabbit RFP (ab185921, Abcam, Cambridge, United Kingdom). Secondary antibodies used were Goat anti-chicken Alexa Fluor 488 (A-11039, Thermo Fisher, Waltham, MA, USA) and Goat anti-rabbit Alexa Fluor 594 (A11007, Thermo Fisher, Waltham, MA, USA). Sections were counter stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma, D9564). After this incubation, sectioned were washed, mounted and coverslipped. Controls performed in parallel without primary antibodies showed very low levels of non-specific staining. Image acquisition was performed with a laser-scanning confocal imaging system (Zeiss LSM710) and image analysis was performed with the ZEN 2009 software (Zeiss, Oberkochen, Germany).