Alexa flour conjugated secondary antibody

Cells grown on coverslip were fixed with the addition of 4% paraformaldehyde (PFA) at room temperature for 20 minutes. Cells were rinsed with PBS and permeabilized by the addition of 0.1% Triton-X100 for 10 minutes at room temperature. Blocking solution (5% BSA in PBS) was added for 1 hour at room temperature. Primary antibody (either anti MPPED2-2B1 mAb (1 μg/ml) or anti-Tuj1 (1:2500, Covance) were diluted to the desired concentration, added to the cells and incubation continued overnight at 4°C. Cells were washed thrice with PBS, and incubated with Alexa Flour-conjugated secondary antibodies (1:200, Invitrogen) for 1 hour at room temperature. Cells were washed thrice with PBS, nuclei stained with Hoechst dye and mounted with anti-fade on glass slides. Coverslips were sealed with nail polish, and confocal imaging performed with a Zeiss LSM 880 confocal microscope with Airyscan detector. Normal mouse IgG, normal rabbit IgG and normal sheep IgG (Sigma) were used as non-specific staining controls.

Brains removed from embryos and pups were fixed for 1 h in phosphate-buffered saline (PBS) containing 4% PFA (w/v), incubated overnight at 4 °C with 20% sucrose in PBS (w/v), embedded in OCT compound (Sakura Finetek, Torrance, CA, USA), and sectioned with a cryostat to obtain 14 µm-thick coronal sections.
For primary antibodies, we used chick antibody to EGFP (Abcam, Cambridge, UK, ab13970), mouse antibody to Rorb (Perseus Proteomics, Tokyo, Japan, N7927), goat antibody to Lhx2 (Santa Cruz, sc-19344), mouse antibody to Brn2 (Santa Cruz, Dallas, TX, USA, sc-393324), and rabbit antibody to Shh (Santa Cruz, c-9024). For some cases, antigen retrieval was performed by incubating the sections for 20 min at 80 °C in 0.01 M sodium citrate buffer (pH 6.0). Because EGFP fluorescence disappeared by the antigen retrieval treatment, EGFP was immunostained with chick antibody against EGFP for revisualization. Immune complexes were detected with Alexa Flour-conjugated secondary antibodies (Invitrogen, Waltham, MA, USA). For nuclear staining, 1 µg/mL Hoechst 33,342 (Invitrogen) was used. Images were acquired using a confocal microscope (SP8, Leica, Wetzlar, Germany).

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature and washed in PBS 3 times, followed by blocking with 5% normal goat serum for 1 hour and incubation with primary antibodies at room temperature (1 hour), washing three times in PBS, and incubation with second antibody for 30 minutes. The following primary antibodies were used for immunostaining: a‐amylase (A8273, rabbit polyclonal, dilution 1:100; Sigma, St. Louis, MO), cytokeratin 19 (CK19) (A53‐B/A2: sc‐6278, mouse monoclonal, dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), insulin (mouse monoclonal, dilution 1:100; Zymed, Waltham, MA), and vimentin (V9: sc‐6260, mouse monoclonal, dilution 1:100; Santa Cruz Biotechnology). The cells were stained with Alexa Flour conjugated secondary antibodies from Invitrogen.

The rabbit anti-GAD65/67 antibody was from Millipore (catalog #AB1511). The mouse anti-GFAP (catalog #3670) antibody was from Cell Signaling Technology. The rabbit anti-GAPDH (catalog #ab181602) antibody was from Abcam. The KA was from Sigma (catalog #K0250). The HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and the Alexa Flour-conjugated secondary antibodies were from Invitrogen. The glutamate receptor antagonists CNQX (catalog #C127) and D-AP5 (catalog #A5282) were purchased from Sigma. The picrotoxin (PTX) was from Abcam (catalog #ab120315). All the other reagents used for preparing electrophysiological recording solutions were purchased from Sigma.

O.C.T. Compound (SAKURA Tissue-Tek^®, Torrance, CA) embedded frozen liver tissues were cut into 5 μm sections. After washed with PBS, sections were fixed with paraformaldehyde, penetrated with Triton X-100 and blocked with bovine serum albumin, primary antibodies anti-α-SMA, anti-EpCAM, anti-laminin, anti-γ-H2AX (all from AbCAM, Cambridge, UK) were probed overnight at 4 °C, proper Alexa Flour conjugated secondary antibodies (Thermo Fisher Scientific) were used to detect the target protein. Images were collected by confocal microscopy (Zeiss LSM710, Oberkochen, Germany).