For dual immunofluorescence staining, tumor sections were deparaffinized, rehydrated, and the antigen was recovered, permeabilized, and blocked with 5% BSA at room temperature for 1 h. The sections were incubated with primary antibodies at 4 °C overnight, followed by Alexa-Fluor conjugated secondary antibodies (Antgene, ANT023, ANT030) at room temperature for 1 h. Mounting was performed using a fluormount containing DAPI (Antgene, ANT063) for further image acquisition (Olympus BX53).
For TUNEL and immunofluorescence co-staining, tumor sections were deparaffinized, rehydrated, and the antigen was recovered, permeabilized, and blocked with 5% BSA for 1 h at room temperature. The sections were stained using a TUNEL assay kit (Vazyme, A112) according to the manufacturer’s instructions and then incubated with primary antibody at 4 °C overnight, followed by incubation with Alexa-Fluor conjugated secondary antibody (Antgene, ANT029) at room temperature for 1 h. Mounting was performed using a fluormount containing DAPI (Antgene, ANT063) for further image acquisition (Olympus BX53).
Quantification of mean fluorescence intensity and TUNEL-positive proportion was performed using ImageJ software. Five sections were assessed in each group, and four randomly selected viewing fields were evaluated individually in each section.
For TUNEL and immunofluorescence co-staining, tumor sections were deparaffinized, rehydrated, and the antigen was recovered, permeabilized, and blocked with 5% BSA for 1 h at room temperature. The sections were stained using a TUNEL assay kit (Vazyme, A112) according to the manufacturer’s instructions and then incubated with primary antibody at 4 °C overnight, followed by incubation with Alexa-Fluor conjugated secondary antibody (Antgene, ANT029) at room temperature for 1 h. Mounting was performed using a fluormount containing DAPI (Antgene, ANT063) for further image acquisition (Olympus BX53).
Quantification of mean fluorescence intensity and TUNEL-positive proportion was performed using ImageJ software. Five sections were assessed in each group, and four randomly selected viewing fields were evaluated individually in each section.