Immunostaining and Quantitative Analysis

Tissue fixation, processing and immunostaining were performed essentially as described [45 (link)]. Tissues were fixed with 4% paraformaldehyde (PFA) in PBS for 1-2 hrs at 4°C, embedded in OCT and cryosectioned at 7-8 μm thickness. Primary antibodies used in this study are listed in Table 3. Secondary antibodies were purchased from Jackson Immunoresearch. To calculate labeling efficiencies, we photographed 5-12 randomly selected 20× fields per stained specimen, across 4-8 sections separated by 100-150 μm. The total number of each cell type (DAPI for total cells per field, LacZ or GFP for Muc1^IC2-labeled cells, insulin and glucagon for endocrine cells, amylase, cytokeratin-19 and DBA lectin for exocrine cells) was determined using the Analyze Particles function of ImageJ (NIH). Double-positive cells were detected by additive image overlay, in ImageJ, of the DAPI channel with lineage⁺ and marker⁺ staining. Accuracy of counts was confirmed by eye in Adobe Photoshop for random samples. Calculations and graphs were generated with Microsoft Excel and R http://www.r-project.org.