Alexa fluor 488 affinipure goat anti rabbit igg

EOC peritoneal MCAs and spheroids induced from cell lines were fixed with 4% paraformaldehyde for 30 min at room temperature and subsequently permeabilized with 0.5% Triton X-100 (Solarbio, China) for 10 min. After blocking with 5% BSA in PBS for 30 min at room temperature, cells were incubated with primary antibodies against EIF5A2 (1:200; Rabbit; Abcam) and CD133 (1:400; Mouse; Proteintech) overnight at 4 °C. Cells were stained with Alexa Fluor 594 AffiniPure goat Anti-Mouse IgG (1:50; A8807110; Yeasen) and Alexa Fluor 488 AffiniPure goat Anti-Rabbit IgG (1:100; A9825370; Yeasen) for 1 h at room temperature in the dark after thorough washing with PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Boster Biological Technology Co Ltd.; Wuhan, China) for 5 min. Immunofluorescence images were observed and collected with a fluorescence microscope (DP72; Olympus; Tokyo, Japan).

The protocol was performed as our laboratory standard. Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated. Blocking of endogenous avidin/biotin, and antigen retrieval was performed by boiling the sections in 0.01 M sodium citrate for 24 min. The sections were then incubated in 0.1% triton for 10 min and blocked with 5% BSA for 60 min, and then incubated with primary antibodies: rabbit anti-human CD54 antibody (CST, Boston, USA), and mouse anti-human ERα antibody (Novus Biologicals, Colorado, USA), at 4°C overnight. Following washing, the tissues sections were incubated with alexa fluor 594-affinipure donkey anti-mouse IgG (Yeasen, Shanghai, China) and alexa fluor 488-affinipure goat anti- rabbit IgG (Yeasen, Shanghai, China) for 60 min at 37°C. Finally, the cell nuclei were stained by DAPI. The results were examined under a fluorescence microscope.