Hrp dab tunel staining kit

Apoptotic cells of paraffin‐embedded tumour sections were detected using the HRP‐DAB TUNEL staining kit (#206386, Abcam, Cambridge, UK) according to the manufacturer's instructions and counterstained with .5% methyl green. Stained slides were imaged under a bright‐field microscope (EVOS XL Core, Invitrogen).

Rat liver tissues were fixed in 10% formalin in paraffin. The samples were sectioned at a thickness of 7 μm. Rat liver tissues were used for the TUNEL assay with an HRP-DAB TUNEL staining kit (Abcam). All stained slides were scanned.

Mice organs and tumor tissues were fixed in 10% formalin, processed, and embedded in paraffin. Four-micrometer tissue sections were deparaffinized, rehydrated, and retrieved by 1 × Trilogy^TM (Sigma-Aldrich, Denmark). Endogenous peroxidase was neutralized by 3% hydrogen peroxide (H₂O₂, Fluka^TM, Germany) for 5 min. After incubation with Protein Block (Leica Biosystems Newcastle Ltd, UK), optimally diluted primary antibodies were added onto tissue sections and incubated overnight at 4 °C, followed by incubation with Post Primary Block and NovoLink Polymer (Leica Biosystems Newcastle Ltd) at room temperature sequentially. The tissue sections were developed by DAB working solution and counterstained with G/M Hematoxylin. After staining, the slides were dehydrated in ascending grades of alcohol, cleared in xylene, and mounted with xylene-based mounting medium (Muto Pure Chemicals Co. Ltd., Japan). Paraffin embedded tumor sections were used for the TUNEL assay by means of the HRP-DAB TUNEL staining kit (Ab206386, Abcam), and the slides were counterstained by methyl blue. All of the stained slides were scanned by histologic digital scanner Pannoramic MIDI (3DHISTECH Ltd, Budapest, Hungary).