Scn400

Immunohistochemical localization was performed with paraffin-embedded tissue sections, deparaffinized and hydrated through xylene and graded alcohols using a standard protocol. Antibodies are used against Caspase 1 (Santa Cruz Biotechnology, Santa Cruz, CA), 8-oxo-dG (Abcam, Cambridge, MA), and Ogg1 (LSBio, Seattle, WA). Appropriate HRP-conjugated secondary antibodies and DAB incubation (Dako North America, Carpinteria, CA, USA) was used for visualization. All the slides were scanned on a Leica SCN400 (Leica Micro System, Buffalo Grove, IL) and analyzed by Tissue IA Optimizer (Leica). The values of positively stained cells were measured in an unbiased manner.

Pancreases were dissected from paired WT and Hid1-betaKO mice and fixed with 4% paraformaldehyde in PBS for 24 hr. After dehydration, the samples were embedded in paraffin. Continuous paraffin sections were obtained at 80 μm intervals from each pancreas. We studied islet morphology for each section using H&E staining, and the sections were scanned with a Leica SCN400 (Leica Microsystems, Wetzlar, Germany). Islet size and number of islets/mm² were determined by ImageJ (National Institutes of Health, https://imagej.nih.gov/ij/ ).
For immunohistochemistry analysis, pancreas slices or cell samples were fixed with 4% paraformaldehyde in PBS for 15 min, followed by permeabilization in PBS containing 0.5% Triton X-100 (MERCK, Billerica, MA) for 10 min and blocking in PBS containing 5% goat serum for 60 min. The samples were incubated for 60 min in PBS containing primary antibodies and 2.5% goat serum and then exposed to fluorescent dye-conjugated secondary antibodies for 60 min at 37°C.

The ablation area was measured using Image J software (version 1.8; National Institutes of Health, Bethesda, Maryland, USA). Adverse events were defined as extensive submucosal hemorrhage, muscularis propria damage, or perforation. Ablation depth was recorded as the deepest histological damaged layer. H&E- and TUNEL-stained tissue specimen slides were analyzed using a slide scanner (Leica SCN400; Leica Microsystems, Wetzlar, Germany) and an image viewer (Leica SCN400 Image Viewer; Leica Microsystems, Wetzlar, Germany).

For the TMA, the whole biopsy was sectioned and stained with Hematoxylin (Histolab Products AB, Sweden). Three representative tumor areas were identified under the light microscope (Olympus BX45, Olympus Corporation, Tokyo, Japan), and three cores of 1,0 mm-diameter were punched with a manual tissue microarrayer (Beecher MTA-1, Estigen,Tartu, Estonia) and re-embedded into a predefined position on a new, empty, paraffin block. The TMA block was heated at 45 °C in 1 h, sectioned, 4 μm, and mounted onto slides.
For IHC analysis, the TMA slides were immunostained by UltraVision Quanto Detection System HRP DAB kit (Thermo Fisher Scientific, Wilmington, DE) and incubated overnight with the MX35 antibody at a concentration of 1:1000. All slides were counterstained with hematoxylin and mounted with Pertex (Histolab Products AB, Sweden). All TMAs were scanned by a Leica SCN400 (Leica Microsystems, Milton Keynes, UK). SlidePath Gateway LAN software was used for the evaluation of the NaPi2b distribution.

Cryosections were analyzed using a laser scanning microscope (LSM 710, Carl Zeiss, Germany) and Zen 2010 black edition (Carl Zeiss, Germany). Alternatively, stained spheroids sections were imaged using a bright field slide scanner (Leica SCN400, Leica Microsystems, Wetzlar, Germany), and images were analyzed using Definiens Tissue Studio 64 software (Definiens AG, Munich, Germany). Photoshop Elements 10 software (Adobe Systems, San José, CA, USA) was used to determine percentages of stained cells and their intensities in spheroid layers.