Immunofluorescence Staining of Colon Cancer TMAs

The tissue microarrays (TMAs) of FFPE colon cancer tissues and normal tissues were purchased from US Biomax Inc. (Rockville, MD). The TMAs contained 155 cases of cancer tissues of different stages and 15 normal tissues, and the clinical pathologic characteristics are listed in Table 1. The FFPE tissue arrays were dewaxed as described previously [23 ,24 (link)]. The TMAs were treated with citrate buffer at pH6.0 (Invitrogen, Grand Island, NY) and then were blocked from non-specific binding by 2% BSA. To achieve double immunofluorescence staining of ALDH1 and other candidate markers, the mouse anti-ALDH1A1 antibody (BD, 1:75) was mixed with the rabbit anti-β-catenin (Abcam, 1:200) and rabbit anti-TGFβ1 (LifeSpan Biosciences, 1:100), respectively, and the mouse anti-NFκB(p65) antibody (Cell signaling, 1:400) was mixed with the rabbit anti-ALDH1A1 antibody (Abcam, 1:200). The antibody mixture was then incubated with the TMAs overnight at 4°C. Then DyLight 549 anti-mouse IgG (red) and DyLight 488 anti-rabbit IgG (green) (Vector laboratories, Burlingame, CA) were diluted (1:200) and incubated with the TMAs for 1 hr at room temperature. Nuclei visualization was performed by DAPI counterstaining (blue) (1:8,000). The TMAs were finally dehydrated in alcohol and cover-slipped.