Computational protocol: Pannexin1 channels dominate ATP release in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing

Similar protocols

Protocol publication

[…] The detailed methods and procedures of immunofluorescent staining can be found in our previous reports. The cochlea was fixed with 4% paraformaldehyde. The cochlear cryostat sections were washed with PBS for 5 min twice and incubated in a blocking solution (10% goat serum and 1% bovine serum albumin) with 0.1% Triton X-100 for 30 min. Then, the tissue sections were incubated with chicken anti-human Panx1 antibody (1:500; #4515, a gift from Dr. Gerhard Dahl at the University of Miami Medical School) in the blocking solution overnight. For double immunofluorescent staining, monoclonal mouse anti-Cx26 antibody (1: 400, Cat# 33-5800, Invitrogen, CA) and polyclonal rabbit anti-Cx30 antibody (1:400, #71-2200, Invitrogen, CA) were used. After being washed with PBS three times, the sections were reacted with corresponding Alexa Fluor 488- or 568 secondary antibodies (1:500, Molecular Probes) for 2 hr at room temperature (23 oC). The staining was observed under a fluorescent microscope or laser confocal microscope. The images were saved in TIFF format for analysis and presentation.For quantitative measurement of Cx26 and C30 labeling at the cochlear lateral wall, ImageJ software (NIH, Bethesda, MD) was used. The density of labeling at the lateral wall was measured and compared between WT and Panx1 cKO mice. [...] Data were expressed as mean ± s.e.m. unless otherwise indicated in text and plotted by SigmaPlot (SPSS Inc. Chicago, IL). The statistical analyses were performed by SPSS v18.0 (SPSS Inc. Chicago, IL) using one-way ANOVA with a Bonferroni correction or t-test. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Deficiency Diseases
Chemicals Adenosine Triphosphate