Dylight 488 anti rabbit igg green

IF double staining protocol was used in order to determine the relative expression of CD90 with CD24 and also the expression difference between CD90 and CD45 or α-SMA. Briefly, the sections were placed in antigen retrieval citrate solution heated at 92–98°C for 15 minutes. Then these sections were blocked with 2% BSA at room temperature. The primary antibodies used were anti-human CD90 with anti-human CD24, mouse anti-human α-SMA (1:400, Sigma) or mouse anti-human CD45 (1:100, Abcam, Cambridge, MA), respectively. These antibodies were added to cover sections and were incubated at 4°C overnight. DyLight 488 anti-rabbit IgG (green) and DyLight 549 anti-mouse IgG (red, Vector laboratories, Burlingame, CA) were incubated (1:150 dilution respectively) in a dark place under normal conditions for 1 hour. Nuclei visualization was performed by incubating DAPI (blue) for 15 minutes. These were washed with PBST for 15 minutes between each step. The sections were finally dehydrated and mounted.

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.

Subclassification of the CD90⁺ population was investigated by the double immunostaining of CD90 with CD24 (CSC marker for PDAC), and cellular markers including α-smooth muscle actin (αSMA, a marker of activated pancreatic stellate cells), CD31 (endothelial vascular cell marker) and CD45 (leukocyte common antigen), respectively. The TMAs were dewaxed, rehydrated, and treated with citrate buffer for antigen retrieval and then 2% BSA to block non-specific binding as described above. To achieve double immunofluorescence (IF) staining of CD90 and the known markers, rabbit anti-CD90 (Abcam, Cambridge, MA) was mixed with mouse anti-CD24 (Abcam, Cambridge, MA), mouse anti-αSMA (Sigma), mouse anti-CD31 (Novocastra, Newcastle Upon Tyne, UK), and mouse anti-CD45 (Abcam, Cambridge, MA) antibodies, respectively, at the optimal dilutions according to the manufacturer's instructions. 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 explored by DAPI counterstaining (blue). The TMAs were finally dehydrated in alcohol and coverslipped.