Mouse anti cd24

Mouse anti-p24, was obtained from NIH AIDS Reagents Program; rabbit antisera to UPF1 and UPF2 were generously supplied by Jens Lykke-Andersen (University of California, San Diego, CA, USA) [34 (link)]; rabbit anti-EST1A (SMG6) and mouse anti-actin were purchased from Abcam; mouse anti-CD24 (henceforth referred as anti-HSA) biotin conjugated clone M1/69 was purchased from BD Biosciences; mouse anti-CD24 (henceforth referred as anti-HSA) PE conjugated clone M1/69 was purchased from eBioscience; rabbit anti-Staufen1 was produced and purified at the McGill University Cell Imaging and Analysis Network (Montréal, Québec, Canada); mouse anti-SAMHD1 was generously supplied by Dr. Mesplède (McGill University) (Abcam); horseradish peroxidase-conjugated secondary antibodies were purchased from Rockland Immunochemicals.

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.