Goat anti doublecortin

Free-floating sections were prewashed three times for 10 min with 0.1 M Tris buffer saline (TBS; Sigma-Aldrich, Oakville, ON, Canada). Sections were then incubated in a primary antibody solution containing 1:500 rabbit anti-zif268 (Santa Cruz Biotechnology, Dallas, TX, USA), 1:500 goat anti-doublecortin (Santa Cruz Biotechnology, Dallas, TX, USA) 0.3% Triton-X (Sigma-Aldrich) and 3% normal donkey serum (NDS; MilliporeSigma, Burlington, MA, USA) in 0.1 M TBS for 24 h at 4 °C. Sections were washed three times for 10 min in TBS and a further incubation of sections commenced in a secondary antibody solution containing 1:500 donkey anti-rabbit ALEXA 594 (Invitrogen, Burlington, ON, Canada), 1:500 donkey anti-goat ALEXA 488 (Invitrogen, Burlington, ON, Canada), 3% NDS and 0.3% Triton-X in 0.1 M TBS for 24 h at 4 °C. Following three final rinses with TBS, the sections were mounted onto microscope slides and cover-slipped with PVA DABCO.

To determine what kinds of cells in the brain AAV-PHP.eB could transduce, sections were stained with rabbit anti-NeuN (GB11138, Servicebio, Wuhan, China), rabbit anti-GFAP (ab7260, abcam, Cambridge, MA, USA), goat anti-Doublecortin (Santa Cruz Biotechnology, Dallas, Texas, USA), and goat anti-CD31 (AF3628, R&D Systems, Minneapolis, MN, USA).
The preparation of sections for immunofluorescent staining was as follows: sections were 1) rinsed in PBS at RT for 5 min; 2) blocked in 5% bovine serum albumin (BSA) in PBS at RT for 1 h; 3) incubated with primary Abs (diluted in 5% BSA) at RT overnight, including the rabbit anti-NeuN Ab (1:100), rabbit anti-Glial fibrillary acidic protein (GFAP) Ab (1:100), goat anti-Doublecortin (DCX) Ab (1:100), and goat anti-CD31 Ab (1:100); 4) washed in PBS, 10 min × 3 times; and, 5) incubated in secondary Abs (diluted in 5% BSA) at RT for 1 h. For primary Abs from rabbit, Alexa Fluor 488-labeled goat anti-rabbit IgG (1:100) were used. Alexa Fluor 488-labeled Donkey anti-goat IgG (1:100) was used for goat primary Ab; 6) washed in PBS, 10 min × 3 times; and, 7) coverslipped using anti-fade mounting medium with or without 10 µg/ml Hoechst dye.

Unused hemispheres from a subset of animals (n = 5) were sectioned coronally on a cryostat at 60 μm and mounted to gelatin-coated slides. Sections were immunostained with mouse anti-nestin (1:100; Millipore), chicken anti-glial fibrillary acidic protein (GFAP; 1:500; Chemicon), goat anti-doublecortin (1:250; Santa Cruz Biotechnology), mouse anti-calretinin (1:200; Millipore) or guinea pig anti-calbindin-d-28K (1:200; Sigma-Aldrich). Alexa Fluor 405 goat anti-mouse, 488 goat anti-chicken, 594 goat anti-mouse, 647 donkey anti-goat or Alexa Fluor 647 goat anti-guinea pig secondary antibodies were used (Invitrogen). Tissue was dehydrated in alcohol series and cleared in xylenes, and coverslips were secured with mounting media (Krystalon, Harleco).

Immunohistochemistry was performed as previously described [44] (link). Briefly, injected animals were perfused at different time points with 4% paraformaldehyde (PFA). The brains were removed and immersed in 4% PFA overnight at 4°C and then equilibrated in 30% sucrose. Entire brains were processed in sagittal 40 microns microtome sections. Sections which were positive with GFP-labelled cells were blocked with 5% donkey serum, permeabilized with 0.25% Triton X in TBS for 30 minutes before application of primary antibodies of rabbit anti-GFAP (Dako, Glostrup, Denmark), anti-PDGFRα (Millipore), goat anti-doublecortin (Santa Cruz, CA, USA), mouse anti-human nestin (Millipore) and anti-human nuclei (Millipore) at dilutions of 1∶100 to 1∶500 for overnight incubation at 4°C. Incubations with secondary antibody (1∶500) of 647 donkey anti-rabbit or 647 donkey anti-goat with 555 donkey anti-mouse for one hour at room temperature were performed, followed by a five minute staining with DAPI (Millipore), before sections were mounted on slides with mounting medium. The staining was viewed using Zeiss LSM 710 confocal system (Carl Zeiss Pte Ltd, Singapore) at 63X magnification.

Samples were fixed with 4% PFA for 10 min at room temperature, subsequently washed with PBS, permeabilized, and blocked in blocking solution (with 2% BSA, 0.1% Triton-X100 in PBS) for 2 h. Primary antibodies were added at 1:100 dilution [rabbit polyclonal anti-MyoVIIa (Proteus); mouse monoclonal anti-Sox2 (Millipore); rabbit polyclonal anti-Sox2 (Invitrogen); rat anti-E-cadherin (Abcam); mouse anti-GATA3 (Thermo Fisher Scientific); mouse anti-Islet 1 (DSHB, deposited by Jessell T.M.); goat anti-Doublecortin (Santa Cruz Biotechnology); rabbit anti-Pax2 (Thermo Fisher Scientific); rabbit anti-Pax8 (Abcam); mouse anti-Nestin (BD Transduction Laboratories); mouse anti-βIII-Tubulin (R&D); rabbit anti-Peripherin (Millipore); and mouse monoclonal anti-Brn3a (Millipore)], and incubated in blocking solution overnight at 4°C. Samples were then washed three times with PBS, followed by the addition of Alexa Fluor conjugated secondary antibodies (Invitrogen) at 1:500 dilution in blocking buffer for 2 days at room temperature. The images were acquired with a confocal microscope (Zeiss LSM 700) using 10× and 20× air objectives.