Dab hrp kit

Following the deparaffinization of the paraffin-embedded osteosarcoma tissue sections, the expression of CT45A1 in the osteosarcoma tissues was detected using a DAB HRP Kit (Beyotime, P0203). Nonspecific binding was blocked by incubating the sections with normal goat-serum for 30 min at room temperature. Endogenous peroxidase activity was quenched by incubating sections in 3% H₂O₂ in PBS for 20 min. Sections were then incubated with anti-CT45A1 monoclonal antibody at 4 °C overnight and washed with PBS (3 × 5 min) before incubating with secondary antibody for 30 min. Slides were washed again (3 × 5 min) with PBS before incubating with the ABC solution. The reaction color was developed by incubating sections with 3,3′-diaminobenzidine reagent. The slides were washed with water and counterstained with hematoxylin. The sections were then dehydrated and mounted with Permount permanent mounting media (Fisher Scientific, Fair Lawn, NJ). All slides were observed under Nikon E400 Light Microscope and representative photographs were taken.

Mouse tumor tissues were immobilized in 4% paraformaldehyde, embedded in paraffin and, cut into 4 μm sections. After deparaffinization, permeabilization, antigen retrieval, and incubation in 3% H₂O₂, tissue sections were incubated with primary antibodies PCNA (1:500, Abcam), PKM2 (1:800; Cell Signaling Technology), PKM1 (1:1000; Cell Signaling Technology), hnRNPA1 (1:100; Abcam). Then, the sections were probed with goat anti-rabbit biotinylated secondary antibody (1:3000, Abcam). Immunoreactive signals were visualized using DAB HRP Kit (Beyotime Biotechnology, Shanghai, China) and the nuclei were counterstained with hematoxylin (Sigma Aldrich).
hematoxylin and eosin (HE) was performed to stain paraffin-embedded tissue sections. Sections were cut into 4 μm slices, and cytoplasm and nuclei were stained with eosin (Sigma Aldrich) and hematoxylin. The images were observed under a microscope.