Ihc 00352

We performed immunohistochemistry as described^{6 (link)}. We applied heart-perfused fixation with 4% paraformaldehyde (PFA). Pancreata were incubated in 4% paraformaldehyde (PFA) at 4 °C overnight, washed with ice-cold PBS three times, and placed in 30% sucrose overnight^{16 (link)}. Tissue was embedded in Tissue-Tek optimal cutting compound (Sakura Finetek), frozen on dry ice, and cut into frozen 5-μm sections. We applied antigen retrieval to detect transcription factors (Nacalai USA Inc.). We used primary antibodies to INSULIN (A056401-2; Dako; 1:1000), GLUCAGON (G2654; Sigma-Aldrich; 1:1000), PDX1 (ab47308; Abcam; 1:100), E-cadherin (61018; BD Biosciences; 1:100), REG1 (AF1657; R&D systems; 1:100), REG2 (AF2035; R&D systems; 1:100), REG3d (MAB5678; R&D systems; 1:100), NR5a2 (PPH2325; R&D systems; 1:100), KI67 (GTX16667; Genetex; 1:100), MAFA (IHC-00352; Bethyl Laboratories; 1:100), Cleaved Caspase-3 (9661; Cell Signaling; 1:100), and ALDH1A3 (NBP2-15339; Novus Biologicals; 1:100) and Alexa Fluor–conjugated goat serum as a secondary antibody (Jackson ImmunoResearch Laboratories and Molecular Probes). The images were captured using a Zeiss LSM 710 confocal microscope using a 20× objective and analyzed using ZEN.

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).