Protocol detail

Quantifying Cholinergic Neuron Markers

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Free-floating basal forebrain sections were processed as previously described.^{29 (link),30 (link)} 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.).

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Vetreno R.P., Bohnsack J.P., Kusumo H., Liu W., Pandey S.C, & Crews F.T. (2019). Neuroimmune and epigenetic involvement in adolescent binge ethanol‐induced loss of basal forebrain cholinergic neurons: Restoration with voluntary exercise. Addiction Biology, 25(2), e12731.

Publication 2019

goat anti-ChAT p75NTR rabbit anti-NeuN mouse anti-BrdU rabbit Alexa Fluor 594 mouse Alexa Fluor 488 goat Alexa Fluor 350 DS-RiZ scope NIS Elements AR46

Alexa 594 Alexa fluor 350 Alexa fluor 488 Anti antibody Antibody cocktail Basal forebrain Brdu Cholinergic neuron Goat Immunofluorescent Mouse P75ntr Rabbit Tissue

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Colocalization of cholinergic neuron markers (ChAT, TrkA, p75NTR)
Colocalization of cholinergic neuron markers (ChAT, NeuN, BrdU)

control variables

None explicitly mentioned

controls

Positive control: Not mentioned
Negative control: Not mentioned

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