Protein lysis and extraction were performed using RIPA lysis buffer. Protein extraction samples were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes, which were incubated with primary coupled antibodies (Table. S1 ) for 12 h at 4 °C. Then, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37 °C. Gel imaging was performed in an iBright CL1500 imaging system (A44240, Thermo Fisher Scientific, USA), and grayscale values were analyzed using ImageJ (version 5.0, Bio-Rad, Hercules, CA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Imagej version 5
Manufactured by Bio-Rad
Sourced in United States
ImageJ (version 5.0) is an open-source image processing software. It provides core functions for viewing, analyzing, and processing digital images. The software supports a variety of image file formats and enables users to perform basic image manipulation tasks such as measuring, enhancing, and processing images.
Automatically generated - may contain errors
2 protocols using imagej version 5
1
Western Blot Protein Analysis
2
Neuron Morphological Identification Assay
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Neuron morphological identification was performed on days 3, 5 and 7 by using an optical microscope (BX53M, Olympus, Japan). After the neurons were incubated for 72 h, they were washed with PBS (0.01 mol/l, pH 7.4) three times, fixed with 4% paraformaldehyde for 20 min, and permeated with 0.1% Effect of miR-1b on neurons via targeting KLF7
TritonX-100 (X100, Sigma-Aldrich, USA) for 30 min. Subsequently, 5% albumin from bovine serum (BSA; #9998, Cell Signaling Technology, USA) was employed for sealing for 20 min. Next, the neurons were incubated with primary antibody anti-MAP-2 antibody (ab183830, 1 : 200, Abcam, UK) overnight at 4%, followed by 1 h incubation with the secondary antibody goat anti-rabbit IgG H&L (ab150077, 1 : 500, Abcam, UK) at 37° without light. Before sealing with glycerine, 1 µg/ml of DAPI staining solution (C1005, Beyotime, China) was added to incubate the neurons for 4 min. The neuron growth states were analysed via ImageJ (version 5.0; Bio-Rad, Hercules, CA, USA).
TritonX-100 (X100, Sigma-Aldrich, USA) for 30 min. Subsequently, 5% albumin from bovine serum (BSA; #9998, Cell Signaling Technology, USA) was employed for sealing for 20 min. Next, the neurons were incubated with primary antibody anti-MAP-2 antibody (ab183830, 1 : 200, Abcam, UK) overnight at 4%, followed by 1 h incubation with the secondary antibody goat anti-rabbit IgG H&L (ab150077, 1 : 500, Abcam, UK) at 37° without light. Before sealing with glycerine, 1 µg/ml of DAPI staining solution (C1005, Beyotime, China) was added to incubate the neurons for 4 min. The neuron growth states were analysed via ImageJ (version 5.0; Bio-Rad, Hercules, CA, USA).
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