Computational protocol: The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

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Protocol publication

[…] Methods for western blot analysis were similar to those reported previously . Briefly, twenty larvae were pooled as one sample (n = 1) and homogenized in a RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride) plus protease inhibitor cocktail (Roche, USA). For western blotting of AQP1a1, extracted protein was heated for 10 min at 70oC, loaded onto a 10% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, USA). The membrane was blocked for 1 h with 5% skimmed milk, probed with eel anti-AQP1a (VNGPDDVPAVEMSSK; 87% identical to the zebrafish AQP1a at the C-terminal) at 1∶1000 dilution in 2% skimmed milk, and then incubated at 4oC overnight. Subsequently, the membrane was probed with 1∶15,000 goat anti-rabbit secondary antibody (Pierce, USA) for 2 h at room temperature, and the immunoreactive bands were detected using Luminata Western HRP Substrates (Millipore, USA). Methods for western blotting with claudin-b antibody have been described elsewhere . To check for equal protein loading, the membrane was re-probed with β-actin antibodies (1∶4000; A2066, Sigma-Aldrich) for 2 h at room temperature after stripping with Re-Blot Plus solution (Millipore, USA). The intensity of the bands was estimated using ImageJ (Rasband 2006, http://imagej.nih.gov/ij/), and the protein expression was normalized to that of β-actin. [...] The time course of water influx was evaluated using non-linear regression analyses:where “Umax” represents the steady state accumulation (equilibrium) of water, “Ku” represents the half-time saturation of water influx, and t is the duration of influx (min).Influx of 3H2O was calculated by:where ‘cpm’ represents counts per minute measured in the fish, ‘SA’ is the specific activity in the water, “n” is the number of fish, and ‘t’ is the duration of the experiment (in minutes for 3H2O influx. Drinking rate was calculated as the slope of a regression line passing through the origin with total incorporated radioactivity of 70kDa [14C]dextran as the dependent variable and flux period (h) as the independent variable.Statistical analyses were performed using Sigmaplot® (version 11.2; SystatSoftware, Inc., USA). All data were analyzed either by Student's t-test or one-way analysis of variance followed by a post-hoc Holm-Sidak test. For the phloretin experiment with AQP1a1 morphants, a two-way ANOVA was performed (morpholino knockdown and phloretin treatment were treated as two independent variables). Data are reported as the mean ± SEM; p<0.05 was taken as the level of significance. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Danio rerio
Chemicals Edetic Acid, Phloretin