Hrp anti goat

Human embryonic kidney 293 T cells were transiently transfected using lipofectamine 3000 (Thermo Fisher Scientific) in Opti-MEM I reduced-serum medium (Thermo Fisher Scientific). Cell lysates and conditioned media were collected after 24 h, and protein expression was monitored by Western blotting using polyclonal goat anti-Cerl2 (R&D), and HRP- anti-goat (Sigma) antibodies. Proteins were visualized using ECL detection reagent (Bio-Rad).

Nitrocellulose membranes were blocked overnight at 4 °C with 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS; in mM 10 Tris, 150 NaCl, pH 7.5, and 0.05% Tween 20®) and then incubated overnight at 4 °C in blocking solution containing the following antibodies: anti-Kir 4.1, anti-AQP-4, anti-COX1, anti-COX2 (diluted 1:1000), and anti-GS (diluted 1:10,000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-actin (1:2000) (Sigma). Subsequently, the membranes were incubated for 1 h at room temperature in a solution containing horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1:10,000), HRP anti-mouse IgG (diluted 1:10,000) (GEHealthcare, Sao Paulo, Brazil), or HRP anti-goat diluted 1:10000 (Sigma). A chemiluminescence signal was detected by luminol substrate reaction (ECL Western Blotting System, GEHealthcare®). Immunoblots were quantified by membrane scanning in an Image4000, GE Healthcare®, and optical densities of proteins studied were determined by ImageJ software (Packard Instrument Company) and the protein/actin ratio calculated.