De-identified specimens from AMD and control subjects were obtained from a tissue repository established in one of our laboratories at Schepens Eye Research Institute under Institutional Review Board approval and in accordance with the ethical standards of the Declaration of Helsinki. Specimens were fixed in 10% buffered formalin, embedded in paraffin, and sectioned. For immunohistochemistry, sections were deparaffinized, and antigen retrieval was performed with citrate buffer (10 mM citric acid, 0.05% Tween-20, pH 6.0, 95–100°C) for 10 minutes. Following two washes in phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO), slides were incubated with a mouse monoclonal anti-human NLRP3 antibody (1:100; clone Nalpy3-b; Enzo Life Sciences, Farmingdale, NY) or a mouse IgG1 isotype control antibody (1:100; Caltag, Carlsbad, CA) overnight at 4°C. The secondary antibody, a biotinylated horse anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA), was visualized via the avidin-biotin-alkaline phosphatase complex (ABC-AP) method (Vectastain ABC-AP Kit; Vector Laboratories) using the Vector Red Substrate (Vector Laboratories). Slides were counterstained with hematoxylin, dehydrated, and mounted with Permount medium (Fisher Scientific, Pittsburgh, PA). For positive controls, human conjunctival tissue corresponding to each eye was also stained with the same antibodies. Uniform expression for NLRP3 was detected (data not shown).