Computational protocol: Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

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Protocol publication

[…] For immunocytochemistry (ICC), cells were differentiated on poly-L-ornithine- and laminin-coated chamber slides (Ibidi). The cells were fixed in Histofix (Histolab AB) for 20 min at room temperature and stained as described previously. Briefly, cells were permeabilized using 0.1% Triton-X100 in DPBS, and thereafter blocked in block buffer (0.1 M glycine, 2% BSA, 0.1% Triton-X100 in PBS). Primary antibodies, TBR2 1:200 (Abcam ab23345), TUJ1 1:1000 (Abcam ab14545), TBR1 1:300 (Abcam ab31940), GFAP 1:1000 (Abcam ab4674), S100 1:400 (Dako Z0311) and VGLUT1 1:750 (Synaptic Systems 135303), were diluted in block buffer and incubated at 4 °C overnight. After washing and re-blocking, samples were incubated with alexa488- and alexa647-conjugated secondary antibodies (Life Technologies) for 2 hours at room temperature and thereafter mounted using Ibidi mounting media (Ibidi).The samples were analysed using a Nikon EclipseTi inverted fluorescent microscope with 10–20× objectives, and images were captured using the DU-897 Andor camera and the Nis Elements software (Nikon). Alternatively, samples were analysed using a Zeiss LSM700 inverted confocal microscope with 40–63× objectives and the ZEN2000 software (Zeiss). Image analysis was performed using ImageJ (NIH). [...] All statistical analyses were performed using the IBM SPSS Statistics 21 software (IBM). Mean values were compared using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis. P-values < 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens