Citra solution

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.).

Paraffin‐embedded postmortem human OFC 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 in a primary antibody solution for 24 h at 4°C. Primary antibodies, dilutions, and validation information are included in Table S2. Slides were incubated for 1 h in biotinylated secondary antibody (1:200; Vector Laboratories) 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 nonspecific binding was conducted employing the above‐mentioned procedures with omission of the antibody.

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. Following incubation in blocking solution (MP Biomedicals), slides were incubated for 24 h at 4°C in a primary antibody solution consisting of blocking solution or Dako antibody diluent (Dako, Denmark) in combination with either rabbit anti‐TLR9 (1:70), rabbit anti‐pNF‐κB p65 (phospho S536) (1:150), mouse anti‐MCP‐1 (1:100), or mouse anti‐IL‐8 (1:300) combined with an antibody against neurons (chicken anti‐NeuN; 1:100; Novus; Cat. #NBP2‐10491), astrocytes (mouse anti‐GFAP; 1:50; Abcam; Cat. #ab4648 or rabbit anti‐GFAP; 1:250; Dako, Denmark; Cat. #Z0334), or microglia (mouse anti‐Iba‐1; 1:250; Wako, Osaka, Japan; Cat. #019‐19741). 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 488 or 594 dye. 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.) and colocalization quantified using NIS Elements AR46 (Nikon Inc.).

Animals were perfused transcardially with PBS solution followed by 4% PFA. Brains were dissected and placed in 4% PFA for 24 h, then block-sectioned and dissected. The 1 cm thick coronal slab which included the injection area (Bregma 1.54 mm) in the center was paraffin-embedded and serially sectioned at 5 μm on a rotary microtome. Slides underwent antigen retrieval (citra solution, BioGenex; 95 °C for 10 min), and were then incubated with 0.3% H₂O₂ to remove the endogenous peroxidase activity. Nonspecific antibody binding was blocked with 3% goat serum in 0.3% Triton-X in PBS for 1 h. Primary antibodies included mouse anti-Cdk5 (1:50, PhosphoSolutions), goat anti-GFP (1:400, Thermo scientific) and rabbit anti-NeuN (1:1000, Millipore). For double labeling, primary antibodies were simultaneously incubated (Cdk5/GFP, cdk5/NeuN). For GFP and NeuN, Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500, Jackson Immunoresearch) were used. For Cdk5, a biotin-conjugated goat anti-mouse IgG (1:2000, Thermo Scientific), followed by streptavidin-HRP and cyanine 3 tyramide (1:50, PerkinElmer), was used. Images were captured using a Zeiss LSM510 Meta confocal laser scanning microscope. Quantitative immunoblot analysis was conducted using previously described standard methodology68 (link).

To assess ChAT colocalization with pNF-κB p65, TLR4, and RAGE, free-floating basal forebrain sections were processed similar to previously reported methods (Vetreno et al., 2019 ). Briefly, sections were washed in 0.1 M Tris-buffered saline (TBS), antigen retrieval performed by incubation in Citra solution (BioGenex, Fremont, CA) for 1 h at 70°C, and blocked with normal horse serum (MP Biomedicals). Sections were incubated for 48 h at 4°C in a primary antibody cocktail of goat anti-ChAT (Millipore) in combination with either mouse anti-TLR4 (Abcam), rabbit anti-RAGE (Abcam), or rabbit anti-pNF-κB p65 (Abcam) (see Table 1). Sections were washed in TBS and incubated for 2 h at room temperature in a secondary antibody cocktail (Alexa Fluor 594, Alexa Fluor 488; Invitrogen, Carlsbad, CA). Tissue was mounted onto slides and cover slipped using Prolong Gold Anti-Fade mounting media (Life Technologies, Grand Island, NY). Immunofluorescent images were obtained using a DS-RiZ scope (Nikon Inc., Melville, NY) and colocalization quantified using NIS Elements AR46 (Nikon Inc.).