Alexa flour 488 and 594 conjugated secondary antibodies

Immunocytochemistry (ICC) of the tumorspheres was performed referring to a previously published protocol [20 (link)]. Briefly, cells were seeded onto poly‐l‐lysine‐coated coverslips and grown in stem cell medium for 3–10 days. After the tumorspheres were formed, cells were fixed in 10% neutral‐buffered formalin (Biosesang, Seongnam, South Korea) for 30 min. Following fixing, the tumorspheres were permeabilized for 5 min and then blocked using 1% BSA (Bovogen, Keilor East, Australia) in PBS (WelGENE Inc.) for 30 min. The cells were then incubated with primary antibodies for 1 h in an incubator maintained at 37 °C. The following primary antibodies were used in this study: OCT4 (1 : 400; Abcam), p62 (1 : 200; Santa Cruz Biotechnology), LC3B (1 : 200; Cell Signaling Technology), and LAMP2 (1 : 200; Santa Cruz Biotechnology). Next, the cells were incubated with Alexa Flour® 488‐ and 594‐conjugated secondary antibodies (1 : 1000; Enzo Life Sciences) for 1 h at room temperature. Lastly, the cells were stained with DAPI‐containing mounting solution (Dako). The cells were visualized using a confocal laser scanning Microscope (LSM 700, Zeiss), and all the images were captured using identical exposure settings. The number of puncta was measured using i‐Solution software (version 22.5, InnerView™). The intensity of nuclear OCT4 fluorescence was measured using imagej [18 (link)].

The tissues processed on a tissue array slide (BC081120f, US Biomax Inc, US Biomax Inc, Rockville, MD, USA) were deparaffinized and rehydrated in HistoClear and serially diluted ethanol (Merck, Darmstadt, Germany). After heat‐induced antigen retrieval, tissues were blocked with normal horse serum for 1 h and incubated overnight at 4 °C with primary antibodies. OCT4 (1 : 400; Abcam) and p62 (1 : 200; Santa Cruz Biotechnology) antibodies were used. Next, nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 15 min and 2 h at room temperature with Alexa Flour® 488‐ and 594‐conjugated secondary antibodies (1 : 1000; Enzo Life Sciences). The slide was mounted with a cover slide using mounting solution (Dako; Agilent Technologies, Santa Clara, CA, USA). Images were obtained with confocal laser scanning microscopy (LSM 700, Zeiss, Oberkochen, Germany) using identical exposure settings. The number of puncta was measured using i‐Solution software (version 22.5, InnerView™, Seongnam, South Korea). The intensity of nuclear OCT4 fluorescence was measured using imagej [18 (link)].