Chicken anti egfp

For immunofluorescent staining, sections were rinsed in PBS and blocked with 5% normal serum/0.1% Triton X-100/PBS at room temperature. Primary antibodies were diluted in blocking solution and incubated 1–2 days at 4°C with gentle agitation. The antibodies utilized were; rabbit anti-Parvalbumin (Swant), chicken anti-EGFP (Aves Labs), rat anti-CTIP2 (Abcam), rabbit anti-SATB2 (Abcam), rabbit anti-CUX1 (Santa Cruz), mouse anti-NEUN (Chemicon), rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology), rabbit anti-IBA1 (Wako), goat anti-IGF1 (R&D Research), rabbit anti-PKCγ (Santa Cruz), rabbit anti-MAP2K1/2(MEK1/2) (Abcam), rabbit anti-P-MAPK1/3(ERK1/2) (Cell Signaling Technology) and rabbit anti-IGF1Rβ (Cell signaling Technology). After rinsing in PBS/T, the secondary antibody was diluted in blocking solution and added overnight at 4°C. Secondary antibodies included Alexa Fluor 488, 546 or 568, and 647 conjugated anti-rabbit, anti-mouse, anti-rat, or anti-goat IgG (Invitrogen). For some experiments slides were then incubated in Hoechst or DAPI for nuclear labeling, rinsed, and mounted. Images were collected with a Zeiss LSM 710, 780, or Leica SP5 laser scanning confocal microscope.

For immunofluorescence labelling of brain sections, injected mice were transcardially perfused with cold phosphate-buffered saline (PBS) for 5 min before switching to PBS containing 4% paraformaldehyde. The whole brains were extracted and postfixed in 4% PFA/PBS for 2 days at 4°C and then cryoprotected in 30% (w/v) sucrose in PBS until they sank, cut horizontally into 40 µm-thick brain sections on a freezing microtome and stored in PBS/50% glycerol at −20°C. For immunostaining, the floating brain sections were washed briefly in PBS and then permeabilized with 0.2% Triton X-100/TBS for 20 min at room temperature, blocked in 5% normal goat serum/PBS for 1 h at room temperature and then incubated with primary antibodies diluted in 3% NGS/PBS overnight at 4°C: chicken anti-eGFP (1:1000, Aves Labs), mouse anti-human tau Tau13 (1:1000, BioLegend) and mouse anti misfolded tau Alz50 (1/100, kind gift from Dr Peter Davis). After washing three times with PBS, secondary antibodies were diluted in 3% NGS/PBS and applied for 1.5 h at room temperature: Alexa 488 anti-chicken, Alexa 555 anti-mouse (1:1000, Thermo Fisher Scientific). After three washes in PBS, sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then mounted on microscope glass slides with mounting media.
Imaging of immunolabelled sections was done using the 40× objective on an Olympus VS120.