Computational protocol: Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson’s disease

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Protocol publication

[…] Cultures were fixed with 4% paraformaldehyde for 30 min at 4 °C, rinsed three times with PBS and blocked for at least 1 h at room temperature with 10% donkey serum in PBS with 0.1% Triton-X (Sigma-Al-drich). Cells were incubated overnight at 4 °C with the following primary antibodies, in blocking solution: anti-aSYN (1:200, sc-7011-R, Santa-Cruz), anti-human aSYN (1:200, sc-12767, Santa-Cruz), anti-β-III-tubulin (1:500, T8660, Sigma-Aldrich), anti-CNPase (1:500, C5922, Sigma-Aldrich), anti-FoxA2 (1:250, sc-6554, Santa-Cruz), anti-GFP (1:500, AB16901, Millipore), anti-Oct4 (1:500, MAB4401, Millipore), anti-TH (1:500, AB152, Millipore; and 1:500, MAB318, Millipore), anti-β-III-tubulin (TUJ1, 1:500, PRB-435P, Covance). After three rinses with PBS, appropriate AlexaFluor-488, AlexaFluor-555 and AlexaFluor-647-labelled secondary antibodies (ThermoFisher Scientific) were used at 1:400 in PBS with 0.1% Triton-X and incubated for 1 h at room temperature in the dark. DAPI (ThermoFisher Scientific) was used for nuclei counterstaining (1:50,000). Images were acquired using an inverted epifluorescent microscope LRI - Olympus IX-73, cell counting was performed using ImageJ or metamorph. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens, Rattus norvegicus
Chemicals Tyrosine