Goat anti mouse

Longitudinal sections of the heart 8 μm thick were cut using a microtome and collected on poly-l-lysine coated slides. After deparaffinization with xylene, the sections were hydrated through graded ethanol. Antigen retrieval was performed with a solution of Tris-EDTA pH 9. Endogenous peroxidase activity in the tissue was eliminated by a 20 min incubation with 3% H₂O₂, and nonspecific binding sites were blocked with BSA 3% in PBS-T for 1 h. The sections were exposed to the following primary antibodies diluted in PBS-T 0.3%: ICAM-1, VCAM-1, PECAM-1, IL1-β, IL-6 (all diluted 1:50), and TNF-α (diluted 1:100), overnight at 4 °C. The product of immune reaction was then revealed by exposing slides for 30 min at 25 °C with the specific biotinylated secondary antibodies: donkey anti-goat, goat anti-rabbit, or goat anti-mouse (BETHYL Laboratories, Inc., Montgomery, TX, USA) diluted 1:200 in PBS-T. The colored reaction product was developed with 3,3′-diamonobenzidine tetrahydrochloride (DAB) solution (Vector Laboratories, Inc., Burlingame, CA, USA). Some sections were incubated with a non-immune serum instead of a primary antibody to assess the background of immunostaining. Before dehydration in ethanol, sections were counterstained with hematoxylin.