Alexa 647 conjugated anti human cd31 antibody

Mouse tumor tissues were dissected from A375SM melanoma and HSC3 oral carcinoma xenografts in nude mice. Human tissue samples were obtained from excised RCC and normal kidney tissues of patients. Tumor specimens embedded in cryocompound (Tissue-Tek; Miles, Elkhart, IN, USA) were immediately immersed in liquid nitrogen and then cut into sections using a cryotome. The frozen sections were fixed in 4% paraformaldehyde for 10 min and then blocked with 2% goat and 5% sheep sera in PBS for 30 min. Mouse sections were double stained with a primary anti-ALDH1A1 antibody (Abcam, Cambridge, UK), Alexa 594-conjugated anti-rabbit IgG (Invitrogen, Tokyo, Japan), and Alexa 647-conjugated anti-mouse CD31 antibody (Biolegend, San Diego, CA, USA). Human sections were double stained with a primary anti-ALDH1A1 antibody (Abcam), Alexa 594-conjugated anti-rabbit IgG (Invitrogen), and Alexa 647-conjugated anti-human CD31 antibody (Biolegend). All immunostained samples were counterstained with DAPI (Roche Diagnostics, Mannheim, Germany) and visualized under a Fluo View FV1000 confocal microscope (Olympus).