Cryosections were air-dried for 30 min and hydrated in Tris-buffered saline (TBS) for 30 min. Glass slides or coverslips were rinsed three times in TBS Triton (0.5%) and incubated in Normal Goat Serum 10%, (Gibco, 16210-064) diluted in Dako Diluent (Dako, S3022) for 30 min. Primary antibodies were incubated overnight in Dako Diluent at 4°C. Chicken anti-EGFP (Invitrogen, A10262, 1:1000), mouse anti-Vimentin (Sigma, V6630, 1:400), mouse anti-NeuN (Millipore, MAB377, 1:100), Rabbit anti Ki67 (Neomarker, clone sp6, RM9106S1, 1:400). After 3 TBS wash, relevant secondary antibodies were incubated in Dako Diluent (Dako, S3022) 1 h at RT, at the following concentrations: Alexa Fluor 488 goat anti-chicken IgY (Invitrogen, A11039, 1:1000), Alexa Fluor 555 goat anti-mouse IgG (Invitrogen, A21422, 1:800), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, A11001, 1:1000), Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen, A21428, 1:800). Nuclear staining was performed using DAPI (Invitrogen, D1306, 3 μM in TBS), for 10 min at RT. Mounting was realized in Fluoromount-G Medium (Southern Biotech, 0100-01).
Confocal examination of the fluorescent labeling was carried out on a LEICA DM 6000 CS SP5 equipped with an Argon laser tuned to 488 nm, a HeNe laser 543 nm, a HeNe laser 633 nm, and a diode 405 nm. Acquisition were performed using oil objectives (×40), thanks to the LAS AF software (Leica).
Confocal examination of the fluorescent labeling was carried out on a LEICA DM 6000 CS SP5 equipped with an Argon laser tuned to 488 nm, a HeNe laser 543 nm, a HeNe laser 633 nm, and a diode 405 nm. Acquisition were performed using oil objectives (×40), thanks to the LAS AF software (Leica).