Nuclear fast red

SA-β-Gal staining was performed in whole-mount tumors or in tumor cryosections preserved in OCT. For whole-mount staining, freshly dissected tumors were cut into 1–3 mm pieces and fixed with a solution containing 2% PFA and 0.2% glutaraldehyde in PBS (pH 6.0) for 2 h on ice. Samples were washed three times with PBS and tissue was incubated overnight in SAβgal staining solution containing X-gal in N-N-dimethylformamide (pH 6.0) at 37 °C. The following day, the samples were washed with PBS then fixed in 4% PFA overnight at 4 °C. Tumor samples were subsenquently dehydrated with two consecutive steps in 50% and 70% ethanol and embedded in paraffin for serial sectioning. 5 μm sections were cut, counterstained with nuclear fast red (Sigma) or were processed for immunohistochemistry as described above with the exception that slides were counterstained for 30 s with nuclear fast red. Detection of SA-β-gal activity in cryosections was performed as previously described63 (link).

It is known that the alcian blue pH 1.0 method primarily detects sulfate groups, and the alcian blue pH 2.5 method preferentially detects carboxyl groups. CWAAPs were stained blue by both staining methods. The present study used the alcian blue pH 1.0 method, which did not stain the mucus of bronchial and alveolar epithelium (Additional file 5: Fig. S5). After deparaffinization and rinsing, the slides were incubated in 0.1 M HCl solution for 3 min. Then, they were incubated in alcian blue staining solution (alcian blue 8GX, C.I.74240, Merck-Millipore, Burlington, MA, USA) for 10 min at room temperature. The slides were then lightly passed through a 0.1N HCl solution and washed with flowing water. Finally, after contrast staining with Kernechtrot (NUCLEAR FAST RED, C.I.60760, Merck-Millipore) for 5 min, the slides were processed for light microscopy.

The precise procedure was described previously^{61 (link)}. Whole mount staining: Mice under anesthesia were perfused through the vascular system with 10 mL of phosphate-buffered saline (PBS) to clear the blood. Perfusion was performed with 10 mL of 4% PFA to obtain adequate fixation. The dissected pancreas of Pdx1-ires-Cre, Rosa LSL-LacZ mice were collected and fixed with ice-cold 4% PFA for 1 h. The tissues were poured into PBS containing 10% of sucrose, and then into PBS containing 20% of sucrose (Nacalai Tesque) for 1 h, respectively, and then placed in PBS containing 30% of sucrose at 4 °C for overnight. Next the pancreas was poured into permeabilization solution (5 mM EGTA, 2 mM MgCl₂, 0.01% sodium deoxycholate, 0.02% Nonidet P-40 in PBS) and reacted with the X-gal solution (1 mg/mL X-gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide in the permeabilization solution) at 37 °C for 6 h. Frozen tissue staining: After fixation of the dissected tissues with PBS containing 30% sucrose, the pancreas was embedded in Tissue-Tek O.C.T compound (Sakura). Frozen tissues were cut serially into 10-μm-thick sections and stained with the procedure described above. Counterstaining was performed using Nuclear Fast Red (Merck Millipore).