Hpa042879

Samples (30 μg) were run out in 10% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked in Tris-buffered saline (10 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 8.00) with 0.05% Tween 20 (TBS-T) containing 5% non-fat dry milk for 1 h at RT. Blots were then incubated overnight at 4 °C with rabbit polyclonal anti-human AQP11 (HPA042879, Sigma) or murine monoclonal anti-β-actin (Sigma) antibodies (diluted 1:1000 and 1:5000, respectively, in blocking solution). The antigen-antibody complexes were visualized using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (diluted 1:5000 in blocking solution) and the enhanced chemiluminescence ECL Plus detection system (Amersham Biosciences, Buckinghamshire, UK). The band intensities were determined by densitometric analysis with the Gel Doc^TM system and Quantity One 4.5.0 software (Bio-Rad) and normalized with β-actin density values.

The indirect immunoperoxidase method was used to detect AQP11 in histological sections of omental and subcutaneous fat [24 (link)]. Sections of formalin-fixed paraffin-embedded adipose tissue (6 µm) were dewaxed in xylene, rehydrated in decreasing concentrations of ethanol and treated with 3% H₂O₂ (Sigma) in absolute methanol for 10 min at RT to quench endogenous peroxidase activity. Slides were blocked during 30 min with 1% murine serum (Sigma) diluted in Tris-buffer saline (TBS) (50 mmol/L Tris, 0.5 mol/L NaCl; pH 7.36) to prevent nonspecific adsorption. Sections were incubated overnight at 4 °C with rabbit polyclonal anti-human AQP11 (HPA042879, Sigma) antibody diluted 1:1000 in TBS. After washing three times (5 min each) with TBS, slides were incubated with DAKO Real^TM EnVision^TM anti-rabbit/mouse (K5007; Dako, Golstrup, Denmark) for 1 h at RT. The peroxidase reaction was visualized using a 0.5 mg/mL diaminobenzidine (DAB)/0.03% H₂O₂ solution diluted in 50 mmol/L Tris-HCl, pH 7.36, and Harris hematoxylin solution (Sigma) as counterstaining. Negative control slides without primary antibody were included to assess nonspecific staining.

Differentiated adipocytes grown on glass coverslips were fixed with 4% formaldehyde for 15 min at RT, incubated with PBS containing 0.3% saponin and 1% BSA for 1 h at RT, and exposed to rabbit polyclonal anti-human AQP11 antibody (HPA042879, Sigma) diluted 1:100 in PBS containing 0.3% saponin and 0.5% BSA overnight at 4 °C. Thereafter, cells were washed with PBS and incubated with Alexa Fluor^® 488-conjugated donkey anti-rabbit IgG (Invitrogen) diluted 1:500 for 2 h at RT. After washing, coverslips were mounted on microscope slides and examined under a TCS-SP2-AOBS confocal laser scanning microscope (Leica Corp., Heidelberg, Germany). After acquisition, images underwent a deconvolution process with the Huygens Essential 2.4.4 software (Scientific Volume Imaging, Hilversum, The Netherlands).