Anti arid1a

Pancreatic specimens were fixed with 10% buffered formalin, dehydrated in ethanol, embedded with paraffin, and stained with hematoxylin and eosin. Standard procedures for IHC analysis were performed according to manufacturer’s protocol. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section. Add enough drops of Hydrogen Peroxide Block to cover the sections. Apply Protein Block and incubate for 5 minutes at room temperature to block nonspecific background staining. Apply primary antibody and incubate according to manufacturer's protocol. Apply Biotinylated goat anti rabbit IgG(H+L) and incubate for 10 minutes at room temperature. Apply Streptavidin Peroxidase and incubate for 10 minutes at room temperature. Add 20ul DAB Chromogen to 1 mL of DAB Substrate, mix by swirling and apply to tissue. Add enough drops of Hematoxylin to cover the section. Rinse 7–8 times in tap water. Add Mounting Medium to cover the section. The image of the IHC stained slides were captured using a Carl Zeiss Axioskop 2 plus microscope (Carl Zeiss, Thornwood, NY). The following primary antibodies were used: Anti-glucagon (Cell Signaling Technology, Cat# 2760); Anti-cytokeratin 19 (GeneTex, GTX112666); Anti-insulin (Cell Signaling Technology, Cat# 3014); Anti-ARID1A (Abnova, MAB15809); Anti-IL-1β (Abcam, ab9722); Anti-F4/80 (Cell Signaling Technology, Cat# 70076).

For Western blot analysis, cells were harvested by RIPA lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris, pH 7.5, 1% NP40, 1% deoxycholate, 0.1% SDS, protease inhibitor cocktail). Cellular proteins were resolved by the 10% Bis-Tris gradient gel (Invitrogen), transferred to the polyvinylidene difluoride membrane, blocked in 5% non-fat milk in PBS/Tween-20, and probed by the following primary antibodies: anti-insulin (Cell Signaling Technology, Cat# 3014); anti-ARID1A (Abnova, MAB15809); anti-Neurogenin3 (Santa Cruz Biotechnology, sc-374442), and anti-MafA (Santa Cruz Biotechnology, sc-390491). Chemiluminescence substrate was applied using SuperSignal West Pico Chemiluminescent Substrate (#34080, Thermo Fisher Scientific) or SuperSignal West Femto Maximum Sensitivity Substrate (#34095, Thermo Fisher Scientific) and blots were analyzed using the ChemiDoc Touch Imaging System (#1708370, Bio-Rad).