Hrp labeled anti ppar α antibody

Immunohistochemistry was performed as described previously [26 (link)]. The tissue sections were incubated at room temperature for 2 h with the following primary antibody: HRP-labeled anti-PPAR-α antibody (1:200, Santa Cruz Biotechnology, catalog no. sc-398394). Slides were rinsed three times with PBS and then stained with DAB using the DAB substrate kit (Nichirei Biosciences). Sections were counterstained with Meyer's hematoxylin, dehydrated, cleared with 99% xylene, and then mounted in malinol. The expression of PPAR-α in cells was observed at 200x magnification using a light microscope (BX53; Olympus Corporation, Tokyo, Japan), and images were captured with a microscopic camera (DP20-5; Olympus Corporation). The classification of nuclear PPAR-α expression was assessed using the following scoring: no staining, 0; <25% positive cells, 1+; 25-50% positive cells, 2+; 50-75% positive cells, 3+; and >75% positive cells, 4+. The expression levels of PPAR-α  were grouped into negative (0, 1+, and 2+) and positive (3+ and 4+) groups.

The cells were seeded on Nunc Lab-Tek chamber slides (Thermo Fisher Scientific) and incubated at 37°C until confluent. Cells were subsequently washed twice with PBS and fixed with 3.7% formalin at room temperature for 15 min. The cells were permeabilized with 0.25% triton in PBS for 10 min and blocked with 2% bovine serum albumin (Wako Pure Chemical Industries) in PBS for 1 h. Cells were incubated at room temperature for 2 h with HRP-labeled anti-PPAR-α antibody (1:200, Santa Cruz Biotechnology, catalog no. sc-398394). The cells were rinsed three times with PBS and then stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB) using a DAB substrate kit (Nichirei Biosciences, Tokyo, Japan). The cells were counterstained with Meyer's hematoxylin, dehydrated, cleared with 99% xylene for 15 min, and mounted in malinol (Muto Pure Chemicals, Tokyo, Japan). The expression of PPAR-α in cells was observed at 200x magnification using a light microscope (BX53; Olympus Corporation, Tokyo, Japan), and images were captured with a microscopic camera (DP20-5; Olympus Corporation).