Computational protocol: Interaction of iPSC-derived neural stem cells on poly(L-lactic acid) nanofibrous scaffolds for possible use in neural tissue engineering

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Protocol publication

[…] For immu-nocytochemistry, cells were fixed with 4% paraformaldehyde for 20 min in 4°C and then washed with phosphate-buffered saline (PBS) three times. After treating with 0.2% Triton X-100 and blocking solution (6% FBS; Life Technologies, Carlsbad, CA, USA) for 1 h in room temperature, the cells were incubated with primary antibodies overnight at 4°C and incubated with the respective secondary antibodies for 1 h at room temperature. Nuclei were detected by Hoechst 33342 (Sigma-Aldrich; Merck KGaA) staining in dark for 4 min at room temperature. Cells were imaged with a fluorescence microscope and confocal laser scanning microscopy (LSM 700; both Leica Microsystems, GmbH, Wetzlar, Germany). Antibodies used targeted: SOX2 (1:400; Abcam, Cambridge, MA, USA), OCT4 (1:400; EMD Millipore), homeobox protein NANOG (NANOG; 1:1,000), stage-specific embryonic antigen 1 (SSEA1; 1:1,000) and NESTiN (1:200) (all from Abcam), microtubule-associated protein 2 (MAP2; 1:800; EMD Millipore), β iii tubulin (Tuj1; 1:400; Abcam), glial fibrillary acidic protein (GFAP; 1:200), myelin basic protein (MBP; 1:200) and paired box 6 protein (PAX6; 1:200) (both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Alexa 488- and Alexa 555-labeled secondary antibodies were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). AP staining was performed using the Alkaline Phosphatase Detection kit (EMD Millipore) according to the manufacturer's protocol. The immunofluorescence was imaged under a confocal laser scanning microscope (LSM 700; Leica Microsystems GmbH, Wetzlar, Germany). Image processing and analysis were realized using ImageJ (National Institutes of Health, Bethesda, MD, USA) and a plugin called NeuronJ (). In this program, we used a computer mouse to select a starting point and an ending point to trace the dendrite in order to get quantification metrics. Data were organized using Excel (Microsoft, Redmond, Washington, USA). […]

Pipeline specifications

Software tools ImageJ, NeuronJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus