Alcian blue ph 2.5 stain kit

Mice pancreata were fixed overnight at 4°C in 10% neutral-buffered formalin, embedded in paraffin, sectioned at a thickness of 3 μm with a Leica RM2245 microtome, and stained with H&E (Sigma). For immunostaining, sections were deparaffinized, rehydrated in a series of ethanol solutions of decreasing concentrations, and subjected to antigen retrieval in sodium citrate buffer (10 mM tri-sodium citrate dehydrate, 0.05% Tween 20, pH 6.0) using a microwave for 15 min. For IHC staining, endogenous peroxidase was inactivated by incubation of sections with 3% H₂O₂ for 10 min. Epitope blocking was performed by incubation of slides with either 10% donkey-serum diluted in PBS containing 0.2% Tween 20 (PBS-T) or 5% bovine albumin serum (BSA) diluted in 0.3% PBS-T for 1h at room temperature. Primary antibodies were incubated overnight at 4°C, followed by secondary antibody incubation for 1 h at room temperature. IHC signals were detected with a DAB peroxidase substrate kit (Vector Laboratories), and images were acquired with a Leica DMLB microscope. Immunofluorescence images were acquired with a Zeiss (LSM 800 or LSM 880) confocal microscope. Nuclei were stained with DAPI (Sigma). Slides were mounted using ProLong Gold anti-fade reagent (Invitrogen). Alcian blue staining was performed using the Alcian blue (pH 2.5) Stain Kit (Vector Laboratories).