Dako realtm envisiontm anti rabbit mouse

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.

The indirect immunoperoxidase method was used to detect AQP7 in histological sections of BAT [16 (link)]. Sections (4 µm) of formalin-fixed, paraffin-embedded BAT 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 for 30 min with 1% murine serum (Sigma) diluted in Tris-buffered 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-AQP7 (sc-28625, Santa Cruz) antibody diluted 1:100 in TBS. After three washes in TBS (5 min each), 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 antibodies were included to assess non-specific staining.