Rabbit alexa fluor 594

HiBCPP cells of BSCFB in vitro model were washed 4 times with PBS or where indicated with acidic PBS (pH = 2.5) after first round of PBS wash, and two times more with PBS afterwards. Cells were fixed with 4% formaldehyde/PBS for 10 min at RT. Filters were subsequently cut out and cells were permeabilized for 10 min with 0.1% Triton X-100/1% BSA/PBS. After washing two times with 1% BSA/PBS, filters were blocked for 20 min in 1% BSA/PBS and stained with primary antibody (anti-ZO-1; 1:250, Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. After washing the filters three times with 1% BSA/PBS the following day, filters were incubated with secondary antibody (anti-rabbit Alexa Fluor 594, 1:250, Invitrogen, Carlsbad, CA, USA) for 2 h at RT and washed three times with 1% BSA/PBS. Cells were incubated with DAPI solution (1.5:55,000, Invitrogen, Carlsbad, CA, USA) to stain the nuclei and with Phalloidin Alexa Fluor 660 (1:250, Invitrogen, Carlsbad, CA, USA) for actin cytoskeleton for 10 min at RT. Filters were washed, mounted with Prolong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) and analyzed by fluorescent microscopy (63× objective, Axio Observer.Z1 with Apotome, ZEN2 pro software, blue edition, Carl Zeiss, Oberkochen, Germany).

Free-floating FSC sections were processed similar to previously reported methods (Vetreno and Crews, 2018 (link); Vetreno et al., 2019 (link)). To assess cholinergic neuron marker colocalization, sections were incubated for 48 h at 4°C in a primary antibody cocktail containing goat anti-ChAT (Millipore), rabbit anti-TrkA (Millipore, Cat. #06-574, RRID:AB_11213262), and mouse anti-nerve growth factor receptor (NGFR; Millipore, Cat. #MAB365, RRID:AB_2152788). To assess ChAT colocalization with phosphorylated (activated) NF-κB p65, FSC sections were incubated for 48 h at 4°C in a primary antibody cocktail containing goat anti-ChAT (Millipore) and rabbit anti-pNF-κB p65 (phospho S536; Abcam, Cat. #ab86299, RRID:AB_1925243). Sections were then washed in TBS and incubated for 2 h at room temperature in the secondary antibody cocktail (Invitrogen; rabbit Alexa Fluor 594 [Cat. #A21207, RRID:AB_141637], mouse Alexa Fluor 488 [Cat. #A21202, RRID:AB_141607], and goat Alexa Fluor 350 [Cat. #A21081, RRID:AB_2535738]). Tissue was mounted onto slides and cover slipped using Prolong Gold Anti-Fade mounting media (Life Technologies, Grand Island, NY, United States). Immunofluorescent images were obtained using a DS-RiZ scope (Nikon Inc., Melville, NY, United States), and colocalization and pNF-κB p65 + IR cells quantified using NIS Elements AR46 (Nikon Inc.).

Free‐floating basal forebrain sections were processed as previously described.29, 30 Briefly, for assessment of cholinergic neuron marker colocalization, sections were incubated for 48 hours at 4°C in a primary antibody cocktail of goat anti‐ChAT (Millipore), TrkA (Millipore), and p75^NTR (Millipore). To assess cholinergic neuron marker colocalization with BrdU, tissue was similarly incubated in a primary antibody cocktail of goat anti‐ChAT (Millipore), rabbit anti‐NeuN (Millipore, Cat. #MABN140), and mouse anti‐BrdU (Millipore). Sections were then incubated for 2 hours at room temperature in the secondary antibody cocktail (rabbit Alexa Fluor 594, mouse Alexa Fluor 488, and goat Alexa Fluor 350; Invitrogen, Carlsbad, CA). Immunofluorescent images were obtained using a DS‐RiZ scope (Nikon Inc., Melville, NY) and colocalization quantified using NIS Elements AR46 (Nikon Inc.).