Nis elements ar46 analysis software

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 human basal forebrain sections were deparaffinized, washed in PBS, and antigen retrieval performed by incubation in Citra solution (BioGenex) for 1 h at 70 °C. Following incubation in blocking solution (MP Biomedicals), slides were incubated for 48 h at 4 °C in a primary antibody solution consisting of Dako antibody diluent (Dako North America) with rabbit anti-REST (Bioss Antibodies) and goat anti-ChAT (Millipore). Slides were washed in PBS and incubated for 1 h at room temperature with Alexa Fluor 488 (Invitrogen, Cat. #A32814) and Alexa Fluor 594 (Invitrogen, Cat. #A21207) secondary antibodies. Secondary-only negative controls were performed without primary antibody incubation. 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.).