Coralite488 conjugated secondary antibody

To visualise the colocalization of the DHFR protein and lysosomes, PLC cells were treated with or without metformin (2.5 mM) for 24 h, and the medium was then replaced with LysoTracker staining buffer (Beyotime, China) for 2 h. Then the cells were washed by PBS twice, fixed in 4% paraformaldehyde, blocked in 2% bovine serum albumin in PBS for 1 h, and incubated with DHFR antibody (1:100) overnight. Then the cells were incubated with Coralite488-conjugated secondary antibody (Proteintech, 1:200) for 2 h, and stained with DAPI (Beyotime) for 10 min. Immunofluorescence images were obtained and analysed using a Zeiss LSM800 microscope and Zen blue software.

The cells were washed with PBS, fixed with 3–4 ml of 4% paraformaldehyde for 30 min at 4 °C, washed with PBS three times and finally permeabilized with a mixture of 10% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and 0.5% Triton X-100 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The cells were immunofluorescently stained with CD31 (Proteintech Group, Inc., Chicago, USA), CD34 (Beijing Bioss Molecular Co., Ltd., Beijing, China), CD133 (Proteintech Group, Inc., Chicago, USA), CD144 (Beijing Bioss Molecular Co., Ltd., Beijing, China) and VEGFR2 (Proteintech Group, Inc., Chicago, USA) antibodies, which were diluted to 100 ml, overnight at 4 °C. The cells were then washed with PBS three times (5 min/time) and incubated with CoraLite 488-conjugated secondary antibody (Proteintech Group, Inc., Chicago, USA) and Alexa Fluor 594-conjugated secondary antibody (Proteintech Group, Inc., Chicago, USA) in the dark at room temperature for 2 h. The cell DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The cells were examined by fluorescence microscopy (IX71, Olympus Corporation, Shibuya, Tokyo, Japan).