Vectorstain elite abc

Briefly, histological sections were subjected to antigen retrieval and peroxidase blocking in 3% H₂O₂ and further blocked in horse serum. Primary antibody (rabbit anti-human Ku80; Cell Signaling Technology, Danvers, MA) was applied and incubated overnight at 4°C. Detection of primary antibody was performed with biotinylated goat anti-rabbit antibodies (Vector Labs). Secondary peroxidase-conjugated antibodies were visualized using a Vectorstain Elite ABC and developed with the VIP kit (Vector Labs). Images were captured with a CKX41 microscope (Olympus, Japan) equipped with a Leica DFC 3200 camera.

For immunohistochemical analysis of mammary tumors, tumors were dissected and fixed in buffered formalin solution for 48 hours. Tissues were embedded in paraffin, and 4-μm sections of the entire block were prepared. After deparaffinization and rehydration, sections were subjected to antigen retrieval in tris/EDTA buffer (pH 8.0) at 120°C for 5 min. Sections were blocked with 1.5% (v/v) horse (Ki67) or goat (p-STAT-1, p-STAT-3, and CD3) serum in PBS for 30 min at room temperature and incubated overnight (4°C) with p-STAT-1 (Tyr⁷⁰¹) (1:500; Cell Signaling Technology), p-STAT-3 (Tyr⁷⁰⁵) (1:200; Cell Signaling Technology), Ki67- (1:400; clone #8D5, Cell Signaling Technology), or CD3-positive cells (1:200; Abcam). After washing with PBS, p-STAT-1, p-STAT-3, Ki67-, or CD3-positive cells were visualized using rabbit (p-STAT-1, p-STAT-3, and CD3) or mouse (Ki67) IgG VECTORSTAIN ABC Elite and DAB Peroxidase Substrate Kits (Vector Laboratories, UK). Sections were counterstained with hematoxylin and imaged on an Olympus CX43 microscope (Olympus, Tokyo, Japan).