Immunohistochemical Staining Protocol for PAS

The tissue sections were stained with an immunohistochemical method for PAS, as previously described [4 (link), 19 (link), 20 (link), 29 (link)]. Briefly, the sections were (1) pretreated with 1:100 proteinase K (Enzo Life Sciences, Farmingdale, NY) diluted in 0.1 mol/L PBS at 37 °C for 20 min; (2) immersed for 30 min in 1% H₂O₂ in 0.1 mol/L PBS with 0.3% Triton X-100 (PBS-TX) at pH 7.4; (3) incubated at 4 °C overnight in anti-PAS monoclonal antibody (psyn no. 64; Wako Richmond, VA) at 1:1000 dilution in PBS-TX; (4) incubated with a secondary biotinylated antibody (anti-mouse IgG diluted 1:1000 in PBS-TX; Vectastain kit, Vector Laboratories, Burlingame, CA) for 2 h at room temperature; (5) treated for 30 min with avidin-biotin complex (Vectastain, Vector Laboratories), with A and B components of the kit both at 1:1000 dilution; and (6) treated with 3,3′-diaminobenzidine (Sigma, St. Louis, MO) (5 mg/100 ml) with added saturated nickel ammonium sulfate (2/100 mL) and H₂O₂ (5 μL/100 mL of 1% H₂O₂) for 30 min in the dark. Controls for staining specificity had no primary antibody.