Neurod1

Organotypic pancreatoids were washed in PBS and fixed in 4% paraformaldehyde for 20 minutes and washed in PBS three times prior to staining or agarose embedding. Tissues were either stained whole mount or embedded into 2% agarose. Tissues were then were blocked for 2 hours room temperature with PBS+ 0.1% Triton X-100 (PBST) with 5% donkey serum (Jackson ImmunoResearch) then incubated overnight at 4 °C in primary antibodies. Tissues were washed in PBST and incubated with secondary antibody overnight at 4 °C. All secondary antibodies were purchased from Jackson ImmunoResearch. Tissues were then washed in PBST before nuclei were stained with Dapi (Invitrogen). Quantification of Insulin+ and Dapi+ cell numbers were performed using ImageJ, with the number of insulin+ cells normalized to the number of Dapi+ cells. Statistics were performed using a Student’s paired t-test.
Antibodies were used as follows: Pdx1 (DSHB at 1:100), Ins (Dako, A056401 at 1:1000), Amy (Sigma, A8273 at 1:400), Chga (DSHB at 1:200), Insm1 (Santa Cruz Biotech. sc-271408 at 1:100), DBA (Vector Lab, RL-1032 at 1:500), Vim (EMD Millipore Corp., Ab5733 at 1:500), Ghrl (Santa Cruz Biotech. SC-10368 at 1:100), Glut2 (Millipore 071–402), Isl1 (DSHB at 1:50), NeuroD1 (Santa Cruz Biotech. Sc-1084 at 1:100), Synaptophysin (Dako M0776 at 1:100), Nkx6-1 (DSHB at 1:100), and MafA (Bethyl Lab. IHC-00352 at 1:100).

The following primary antibodies were used in the study; β-Catenin (1:200, BD Biosciences, NC USA), Phosphohistone H3 (PH3) (1:100, Cell Signaling, Boston, MA USA), PCNA (1:100, Cell Signaling), N-Cadherin (1:200, BD Biosciences), NeuroD1 (1:100, Santa Cruz, Dallas, TX USA), β-III Tubulin (1:2000, Promega, Fitchburg, WI USA; 1:3000, Covance, Princeton, NJ USA), Shh (1:100, Santa Cruz), Wnt3a (1:100, Santa Cruz) and Cell Mask Deep Red Plasma membrane stain (1:500, Molecular Probes, Grand Island, NY USA). Alexa Fluor anti-rabbit and anti-mouse 488 and 594 secondary antibodies were used at a dilution of 1:1000 (Molecular Probes). Sections from control, cyclopamine and SAG treated mice were stained with Nissl stain. The protocols for IHC and Nissl staining have been described previously (Haldipur et al., 2012 (link)).

Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then processed for immunocytochemistry [13 (link)]. Samples were rinsed with PBS three times. Then, samples were incubated at 4°C overnight with the primary antibodies diluted in PBS containing 5% of fetal bovine serum and 0.3% Triton X-100. The primary antibodies used were as follows; Synaptophysin (1:50000, Millipore, USA), MAP2 (1:1000, Sigma-Aldrich, USA), MAP2 (1:500, Millipore, USA), vGlut1 (1:2000, Synaptic systems, Germany), GABA (1:1000, Sigma-Aldrich, USA), PSD95 (1:500, Millipore, USA), α-SMA (1:500, Sigma-Aldrich, USA), Sox2 (1:500, R&D, USA), Ascl1 (1:200, BD, USA), Brn2 (1:200, Santa Cruz, USA), Myt1L (1:500, Abcam, England) and NeuroD1 (1:500, Santa Cruz, USA). After three washes with PBS, samples were incubated with secondary antibodies conjugated with Alexa-488, Alexa-555 and Alexa-647 (Invitrogen, USA). Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI; 1:1000, Dojindo, Japan). After washing with PBS, samples were mounted on slides with FluorSave reagent (Calbiochem, Germany) and examined under a universal fluorescence microscope (Axioplan 2; Carl Zeiss, Germany). For anti-BrdU staining (1:500, Abcam, England), cells were treated with 1 N HCl in PBS for 30 minutes at 37°C and then rinsed with PBS three times before primary antibody incubation.

N39 cells were seeded on poly-l-lysine–coated coverslips. After treatment with Wnt3a for 24 h, cells were washed with PBS, fixed with 4 % paraformaldehyde and permeabilized in 0.2 % Triton X-100, 0.1 M glycine. The cells were then washed with PBS, blocked with BSA containing antibody diluent (Invitrogen) at room temperature (RT) and incubated with antibodies against proinsulin (1:50; R&D Systems), non-phospho β-catenin (1:500; Cell Signaling), and NeuroD1 (1:200; Santa Cruz) at 4 °C. Coverslips were incubated with secondary antibodies for 2 h at RT, and Hoechst 33342 (Invitrogen) was used for nuclear staining for 10 min at RT. ProLong Diamond Antifade Mountant (Invitrogen) was used for mounting coverslips. Each slide was analyzed with an LSM700 confocal microscope (Carl Zeiss). Images were analyzed with ZEN2012 software (Carl Zeiss).