Computational protocol: Developmental abnormalities in supporting cell phalangeal processes and cytoskeleton in the Gjb2 knockdown mouse model

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Protocol publication

[…] Eighteen days after TMX injection, mice were deeply anesthetized by i.p. injection with a combination of ketamine and xylazine, and the cochleae were carefully dissected from the temporal bones and fixed in 4% paraformaldehyde in 0.01 M PBS at room temperature for 1 h. The apical stretched preparation was carefully dissected from freshly dissected cochleae in 0.01 M PBS. The flattened cochlear preparations were incubated in a blocking solution (10% donkey serum with 0.1% Triton X-100) for 1 h at room temperature. The tissue was then incubated with polyclonal rabbit anti-Cx26 antibodies (1:200 dilution; 512800; Invitrogen), polyclonal rabbit anti-myosin7a antibody (1:500 dilution; 25-6790; Proteus BioSciences, USA), monoclonal rabbit anti-α-tubulin antibodies (1:200 dilution; ab179484; Abcam, UK), and polyclonal goat anti-sox2 antibodies (1:100 dilution; sc-17320; Santa Cruz Biotechnology, USA) diluted in 0.01 M PBS with 0.3% Triton X-100 overnight at 4°C. Tissues were washed in 0.01 M PBS with 0.1% Tween-20 and were stained by Alexa Fluor 647 donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-goat IgG (1:200 dilution; ANT032 and ANT031; antgene, PR China) for 1 h. DAPI (C1005; Beyotime Biotechnology) and phalloidin (0.05 mg/ml; P5282; Sigma, USA) were used for nucleus and F-actin staining, respectively. Images were obtained with a laser scanning confocal microscope (Nikon, Japan). The distance between the nuclei of the IPCs and outer OPCs was measured with Image-Pro Plus 6.0, and the three-dimensional reconstruction images were produced with the NIS-Element software (Nikon, Japan). The immunolabeling of acetylated α-tubulin was quantified from original images, each taken at ×60 magnification in identical conditions. The images were analyzed with ImageJ software, and the relative fluorescence was quantified by normalizing the ratio of average fluorescence of PCs in the KD group (n=3) to the average fluorescence of PCs in the corresponding control group (n=3). [...] All data are presented as means±s.e.m. and plotted using Sigma Plot (Version 12.5; Systat Software, Inc., USA). One-way ANOVA with LSD correction or t-tests were performed in SPSS software (version 19; IBM SPSS Statistics, USA), and P<0.05 was considered to be statistically significant. […]

Pipeline specifications

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