Goat alexa fluor 350

To assess cholinergic neuron marker colocalization, free-floating basal forebrain sections were processed similar to previously reported methods [32 (link), 33 (link)]. Briefly, sections collected from subjects sacrificed on P80 were washed in 0.1 M Tris-buffered saline (TBS), antigen retrieval performed by incubation in Citra solution (BioGenex, Fremont, CA) for one hr at 70°C, and blocked with normal horse serum (MP Biomedicals). Sections were incubated for 48 hr at 4°C in a primary antibody cocktail of goat anti-choline acetyltransferase (ChAT; Millipore) with an antibody against TrkA (rabbit anti-TrkA [Millipore]) and p75^NTR (mouse anti-p75^NTR [Millipore]). Sections were then washed in TBS and incubated for 2 hr at room temperature in the secondary antibody cocktail (rabbit Alexa Fluor 594, mouse Alexa Fluor 488, and goat Alexa Fluor 350; 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.).

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.).