Rat anti e cadherin

Whole mount immunostaining of embryonic lungs was performed as previously described [36 (link)]. Briefly, the whole embryonic lungs were dissected out and fixed in 4% PFA on ice for 1 h. Lungs were washed with PBS and dehydrated in graded methanols (25%, 50%, 75%, 100%). After incubating in 5% H₂O₂/methanol for 4 h, the samples were then rehydrated through graded methanols (100%, 75%, 50%, 25%, 0%) diluted in 0.1% Tween-20/PBS and incubated with blocking buffer (1.5% BSA/0.5% Triton X-100/PBS) for 2 h. The samples were then incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: rat anti-E cadherin (1:200, Life Technologies, Cat# 13–1900), goat anti-SOX9 (1:100, R&D Systems, Cat# AF3075, RRID:AB_2194160), rabbit anti-FAK (1:100, Cell Signaling Technology, Cat# 3285S, RRID:AB_2269034), and rabbit anti-Phospho-FAK (Tyr397) (1:100, Cell Signaling Technology, Cat# 3283S, RRID:AB_2173659). On the second day, the samples were washed with blocking buffer for 5 h, then incubated with secondary antibodies at 4°C overnight. The final day, the samples were washed for 5 h with blocking buffer. Images were captured using a Nikon Eclipse E1000 microscope with SPOT 2.3 CCD camera.

Whole-mount immunostaining was performed as previously described^{48 ,49 (link)}. Briefly, the embryonic lungs at the indicated time points were collected and fixed in 4% PFA on ice for 1 h. Samples were washed with 0.1% Tween-20/PBS for 30 min then dehydrated in graded methanol solutions (25%, 50%, 75%, 100%). After incubating in 5% H₂O₂/methanol overnight, the lungs were rehydrated through graded methanol solutions (100%, 75%, 50%, 25%, 0%) which diluted in 0.1% Tween-20/PBS and incubated in blocking buffer (1.5% BSA/0.5% Triton X-100/PBS) for 6 h. Primary antibodies were incubated at 4 °C overnight. On the next day, lungs were washed with blocking buffer for 5 h, then incubated with secondary antibodies at 4 °C overnight. Images were captured on a Nikon Eclipse E1000 microscope with a SPOT 2.3 CCD camera. The primary antibodies used were: rat anti-E Cadherin (1:200, Life Technologies, Cat# 13-1900; RRID:AB_2533005), rabbit anti-SOX2 (D9B8N) (1:200, Cell Signaling Technology, Cat# 23064; RRID:AB_2714146), goat anti-SOX9 (1:200, R&D Systems, Cat# AF3075; RRID:AB_2194160), rabbit anti-prosurfactant protein C (proSP-C) (1:200, MilliporeSigma, Cat# AB3786; RRID:AB_91588), rat anti-PECAM-1 (CD31) (1:150, Santa Cruz Biotechnology, Cat# sc-18916; RRID:AB_627028).

The following primary antibodies were used in this study: Rabbit anti-dsRed (Clontech #632496), Rat anti-E Cadherin (Life Technologies #13–1900), Rabbit anti-Fat3 and polyclonal Mouse anti-Fat3 (Lisa Goodrich, Harvard Medical School [18 (link)]), Goat anti-GFP (Abcam #5450, IF and WB), Goat anti-GFP (Abcam #6673, IP only), Rabbit anti-GST (Cell Signaling Technology #2625S) Mouse anti-HA (Covance #MMS-101P), Mouse anti-Kif1A (BD Biosciences #612094), Rabbit anti-Kif5B (Thermo # PA1-642), Goat anti-Kif5B (Imgenex #IMG-3049), Rabbit anti-Kif5C (Acris # SP5236P), Mouse anti-Phospho-Tyrosine (Cell Signaling Technology #9411). Fluorescent, Dylight conjugated secondary antibodies were purchased from Jackson Immunoresearch, and HRP conjugated secondary antibodies used for Western blot were from BioRad. Whole IgG negative controls for IP experiments were from Sino Biological (Goat IgG #CR2-500) and Millipore (Mouse IgG #12–371).

Cell culture reagents were purchased from Mediatech (Manassas, VA). General chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The following primary antibodies were used: mouse anti-podocalyxin was a gift from Dr. Karl Matlin (University of Chicago); rabbit anti-PKCζ (Santa Cruz Biotechnology, Dallas, TX); mouse anti-tubulin, mouse anti-β-actin, rhodamine-phalloidin, rat anti-E-cadherin (Sigma), chicken anti-GFP (Life Technologies, Grand Island, NY); rabbit anti-NHERF1 (Abcam, Cambridge, MA); rabbit anti-Par3 (EMD Millipore, Billerica, MA); rabbit anti-cingulin was a gift from Dr. Rytis Prekeris (University of Colorado, Denver). Secondary antibodies were purchased from Molecular Probes/Thermo-Fisher (Eugene, OR). GFP-Cdc42 and GFP-Cdc42Q61L were purchased from Addgene (Cambridge, MA). GFP-PH-PLCδ, GFP-PH-Akt, and Annexin2-GFP plasmids were gifts from Dr. Keith Mostov (University of California, San Francisco). Arf6-GFP and PtdIns(4)P 5 K plasmids were described previously85 86 (link). GFP-Rab11a, GFP-Rab14, and GFP-Rab14S25N were prepared as described48 . Matrigel was purchased from Corning Life Sciences (Corning, NY).

For measurement of endocrine-cell mass, a minimum of 12 pancreas sections spanning the entire pancreas were assessed for at least 3 different mice per genotype. The total cross-sectional area of hormone⁺ cells was summed and normalized to total pancreatic area using Image-Pro Plus analysis software (Media Cybernetics). Statistical analysis was performed using a two-tailed Student's t-test. For staining Runx1t1 and Etv1-LacZ expression, E15 and 2-month old mouse pancreata were dissected and fixed with 4% paraformaldehyde overnight at 4°C, and cryo-embedded. Sections were permeabilized with 1% Triton-X-100 for 1 hr before blocking with 2%BSA, 1% DMSO in PBS. We used the following primary antibodies: Goat anti-Runx1t1 (1∶200, Santa Cruz, C-20), Rabbit anti-LacZ (1∶500, Invitrogen), and Rat anti-E-cadherin (1∶400, Invitrogen). Secondary antibodies were from Jackson ImmunoResearch and Molecular Probes. Samples were mounted with Vectashield containing DAPI (Vector Laboratories). Microscopic images were obtained using a Leica SP2 AOBS confocal laser-scanning microscope.