Goat anti mouse igg p0448

To detect kidney injury, the fixed right kidney was dehydrated in ethanol and embedded in paraffin. Kidney tissue blocks were cut into 2-μm-thick sections and subjected to hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and Masson’s trichrome staining. For immunohistochemical analysis of kidney tissues, we used the following antibodies: mouse monoclonal against CD68 (ED1; 1:100; Abcam, Cambridge, MA, USA) and rabbit polyclonal against tumor necrosis factor-α (TNF-α; 1:200; Abcam). Next, secondary antibody was performed using HRP-conjugated polyclonal goat anti-rabbit IgG P0447 or goat anti-mouse IgG p0448 (Dako, Glostrup, Denmark) for 1 h. The sections were visualized using 3,3-diaminobenzidine (DAB; DAKO ChemMate Detection Kit) and counterstained with Mayer’s hematoxylin.

Protein concentration was measured using Bradford’s method in lysates of treated HK2 cells and tissues. Total protein (20 µg) was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 10% skim milk for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies. The membrane was incubated with HRP-conjugated polyclonal goat anti-rabbit IgG P0447 or goat anti-mouse IgG p0448 (Dako, Glostrup, Denmark) as the secondary antibody for 1 h and detected using advanced ECL reagents (Amersham Bioscience, Piscataway, NJ, USA). The target protein bands were normalized to that of GAPDH. Expression levels were estimated using Scion Image software (Scion, Frederick, MD, USA). The primary antibodies that detect proteins are listed in Table 2.