Diaminobenzidine chromogen

Avidin-biotin-peroxidase was employed to determine apoptotic cells in myocardial
tissue. Sections 2-3 µm in thickness taken from the myocardial tissue
paraffin blocks were placed onto positively charged slides. These sections were
subsequently deparaffinized by being stored for 15 minutes in 3% H₂O₂ solution.
A blocking solution was then applied for 20 seconds, after which the sections
were incubated first with primary antibody (Caspase-3, Rabbit polyclonal, Abcam,
United Kingdom) and then with secondary antibody (Goat Anti-Rabbit IgG H&L
[^HRP]) (ab205718, Abcam, United Kingdom) for 60 minutes. After
being kept in diaminobenzidine chromogen (DAB Chromogen, Abcam, United Kingdom)
solution for 15 minutes, the tissues were then counterstained with Harris
hematoxylin (Merck, Darmstadt, Germany) and covered with an appropriate
solution.

Expression of two markers, Ki67 and F4/80, was evaluated using standard methods for immunohistochemistry and commercial antibodies including rabbit anti-Ki67 (Fisher Scientific, Cat# RM-9106-S1), a marker of proliferation, and F4/80 (a macrophage marker). Briefly, sections were deparaffinized, hydrated through a series of alcohols, microwaved in 10 mM citrate buffer (pH 6) for antigen retrieval, and treated with hydrogen peroxide to quench endogenous peroxidases. Non-specific binding was blocked with 1% milk protein in 5% normal goal serum (Cell Signaling Technology, Danvers, MA).
Sections were incubated with primary antibodies (typically diluted 1:500 or 1:1000 in blocking solution) at 4°C for 14–16 h. They were then washed and incubated with secondary antibody (goat anti-rabbit, Abcam, Cat# ab64256, undiluted) followed by streptavidin peroxidase complex (Abcam, Cat# ab64269). Diaminobenzidine chromogen (Abcam, Cat# ab64238) was used to visualize reactions. Sections were counterstained with hematoxylin (Fisher Scientific). Each immunohistochemical run included a negative control in which the primary antibody was replaced with 5% normal goat serum.