Elite avidin biotin enzyme complex abc

Immunohistochemical (IHC) labeling on adjacent tissue sections was performed to identify ferritin protein expression (rabbit anti-H-ferritin or rabbit anti-L-ferritin; Abcam). Briefly, sections were rinsed in 0.1 M PBS, blocked for nonspecific antigen binding using 4%BSA/0.3% Triton-100/PBS for 1 h and then incubated with primary antibodies overnight at 4 °C. The next day, sections were rinsed and incubated with secondary antibodies (goat anti-rabbit IgG; Vector) for 1 h. After rinsing, endogenous peroxidase activity was quenched using a 4:1 solution of methanol/30% hydrogen peroxide for 15 min in the dark. Sections were treated with Elite avidin– biotin enzyme complex (ABC; Vector Laboratories) for 1 h. Visualization of labeling was achieved using DAB substrates (Vector Laboratories). Sections were rinsed, dehydrated, and coverslipped with Permount (Thermo Fisher Scientific). All slides were imaged using a Zeiss Axioshop 2 Plus microscope equipped with a Sony 970 three-chip colored camera.

Sections were rinsed in 0.1M PBS, then endogenous peroxidase activity was blocked using 10:1 methanol/30% hydrogen peroxide for 20 min in the dark. After rinsing with PBS, sections were blocked for nonspecific antigen binding using 4% BSA/0.3% Triton X-100/PBS (BP³⁺) for 1 h in a humid chamber. Primary antibodies (Table 1) were diluted in BP³⁺ and applied to sections overnight at room temperature. Next, sections were rinsed and treated with biotinylated secondary antibodies for 1 h at room temperature. Sections were primed with Elite avidin-biotin enzyme complex (ABC; Vector Laboratories) for 1 h, and labeling was visualized with ImmPACT^TM DAB (Vector Laboratories). Sections were rinsed, dehydrated, and coverslipped with Permount (Thermo Fisher Scientific).