Immunofluorescence Staining of CD90 and CD24

Protein expression was determined by immunofluoresence (IF) staining [7 (link)]. Briefly, the tissue specimens were placed in xylene twice for 20 minutes and hydrated in 100% ethanol, 95% ethanol, 75% ethanol, 5 minutes each and water for 3 minutes. Antigen retrieval was completed by boiling the sections in solution (BioGenex HK080-9K) for 15 minutes. Subsequently, these were blocked with 2% BSA in PBS containing 0.05% Tween (PBST) for 1 hour at room temperature and incubated with rabbit anti-human CD90 antibody (1:100, Abcam, Cambridge, MA) or mouse anti-human CD24 antibody (1:150, Abcam, Cambridge, MA) at 4°C overnight. The secondary antibodies of DyLight 488 anti-rabbit IgG (green) or DyLight 549 anti-mouse IgG (red, Vector Laboratories, Burlingame, CA) were used at 1:150 dilution and incubated in a dark place for 1 hour at room temperature. CD90 staining was visualized in green and CD24 in red. Staining with DAPI (blue, 1:4000 dilution) presented nuclei visualization. The sections were dehydrated in alcohol and xylene, then cover-slipped with TM mounting medium (Sigma, USA).