Alexa fluor 488 conjugated goat anti mouse antibody

Coverslips were coated with 0.5% gelatin (Sigma-Aldrich, St. Louis, USA) and three different cell lines of prostate cancer were seeded onto different coverslips and cultured in RPMI-1640 medium supplemented with 10% FBS. When cells grew to 70% confluence on the coverslips, they were fixed with 4% paraformaldehyde for 30 min and incubated with 0.2% Triton X-100 for 5 min at 25°C. The cells were then treated with 10% normal goat serum (Abcam, ab7481), and incubated with primary mouse anti-human antibodies against CD59, Haptoglobin, and Tetranectin (all from Abcam,the same catalog number as immunohistochemistry) at a dilution of 1:100 at 4°C overnight, followed by Alexa Fluor® 488-conjugated goat anti-mouse antibody at a dilution of 1:200 (Abcam, ab150113) at 25°C for 1 h in the dark. After staining with DAPI (CST, Danvers, USA) for 5 min to visualize the nuclei, cells were imaged by fluorescence microscopy (Olympus, Japan).

Western blotting and IF were performed to evaluate the specific protein expression as described previously (Han et al., 2020a (link)). In addition to the above-mentioned primary antibodies for IHC staining, HIF-1α (610958, BD Biosciences, New Jersey, United States) for WB was also used. Secondary antibodies included anti-rabbit or anti-mouse HRP-linked antibody (Cell Signaling Technology, MA, United States), Alexa Fluor 488^®-conjugated goat anti-mouse antibody (Abcam), Alexa Fluor 594^®-conjugated goat anti-rabbit antibody (Abcam). The protein samples of siControl and siHIF-1α SHED were collected after culturing under normoxia and hypoxia for 24 h. The results of WB were quantified by Image J and normalized by Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells seeded on coverslips in 24-well plates were fixed by 4% PFA after incubated in normoxia and hypoxia for IF staining. After incubated with primary antibody overnight, secondary antibody for 1 h and DAPI for 5 min, images were captured by fluorescence microscope (Nikon, Tokyo, Japan) at 20 × magnification.