Blocking solution

Paraffin‐embedded human OFC sections were deparaffinized, washed in PBS, and antigen retrieval performed by incubation in Citra solution (BioGenex) for 1 h at 70°C. For double immunofluorescent immunohistochemistry, slides were incubated in blocking solution (MP Biomedicals) and incubated for 24 h at 4°C in a primary antibody solution consisting of blocking solution with NOX2 in combination with an antibody against either GRP78, ATF6, CHOP, cleaved caspase 3, or caspase 12 (Table 3). For triple immunofluorescent immunohistochemistry, slides were incubated for 24 h at 4°C in a primary antibody solution consisting of blocking solution with cleaved caspase 3 and NeuN in combination with an antibody against either NOX2, GRP78, or ATF6. Slides were washed in PBS and incubated for 1 h at room temperature with the appropriate secondary antibodies (1:1000). Immunoblotting was visualized using Alexa Fluor 350, 488 or 594 dye as necessary. Secondary‐only negative controls were performed without primary antibody incubation. Slides were coverslipped using Prolong Gold Anti‐fade mounting media (Life Technologies). Immunofluorescent images were obtained using a Nikon DS‐Ri2 scope (Nikon Inc., Melville, NY) and colocalization quantified using NIS Elements AR46 analysis software (Nikon Inc.).

Paraffin‐embedded post‐mortem human OFC sections were deparaffinized, washed in PBS, and antigen retrieval performed by incubation in Citra solution (BioGenex, San Ramon, CA) for 1 h at 70°C. Following incubation in blocking solution (MP Biomedicals, Solon, OH), slides were incubated in a primary antibody solution for 24 h at 4°C. Primary antibodies, dilutions, and validation information are included in Table 3. Slides were incubated for 1 h in biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) and then for 1 h in avidin–biotin complex solution (Vector Laboratories). The chromogen nickel‐enhanced diaminobenzidine (Sigma‐Aldrich) was used to visualize immunoreactivity. Slides were dehydrated and cover slipped. Negative control for non‐specific binding was conducted employing the above‐mentioned procedures with omission of the antibody.