TdT-mediated dUTP nick end labeling (TUNEL) and Ki67 staining at the end of the 16-day experimental period were used to analyze the apoptosis and proliferation of the tumor tissues. The tumor tissue was removed, fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin for preservation. Paraffin sections were dewaxed with xylene, dehydrated with gradient alcohol, subjected to antigen retrieval, blocked and then incubated with antibodies against Ki67 overnight. After the appropriate specific secondary antibody was added, the staining results were observed under a microscope after the color development of DAB.
Paraffin sections were dewaxed with xylene, dehydrated with gradient alcohol and washed with PBS. Proteinase K buffer (Sigma, USA) was added for 30 min. After washing with PBS, 50 μL TUNEL reaction mixture (5 μL TdT+45 μL fluorescein labeled dUTP solution) was added, and the slides were placed in a humidified box at 37° in the dark for 1 h. The slides were washed with PBS and incubated with DAPI for 5 min. Then, the slices were sealed and observed under a fluorescence microscope.
Paraffin sections were dewaxed with xylene, dehydrated with gradient alcohol and washed with PBS. Proteinase K buffer (Sigma, USA) was added for 30 min. After washing with PBS, 50 μL TUNEL reaction mixture (5 μL TdT+45 μL fluorescein labeled dUTP solution) was added, and the slides were placed in a humidified box at 37° in the dark for 1 h. The slides were washed with PBS and incubated with DAPI for 5 min. Then, the slices were sealed and observed under a fluorescence microscope.