Anti-CD133 antibody - Stem Cell Marker (ab19898)

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"The tumors were removed and fixed in 10% formalin. Following tissue preparation, paraffin blocks were sectioned using a Leitz 15/2 microtome. Based on the standard IHC protocol, mouse monoclonal antibody was used to stain tumor tissue samples against CD133 (Cat # ab16518; Abcam) and NF-kB (Cat # sc-8008; Santa-Cruz). Next, xylene was used to deparaffinize paraffin-embedded Sects. (5 µm). Also, 3% H2O2 was used to block endogenous peroxidase activity in methanol for half an hour. After dehydrating the tissue sections with graded alcohol, antigen retrieval was achieved using 10 mM sodium citrate. Next, TBST was used to wash the sections, while 5% bovine serum albumin (BSA) was used for blocking in 1 h.

Slides were incubated with the mouse monoclonal antibody against NF-kB and CD133 (diluted in TBS at 1∶50). After washing the slides in TBST for 5 min, they were incubated with HRP-conjugated anti-mouse antibody, which was diluted in TBS (1∶200). The slides were incubated with DAB (Sigma) after TBS washing; following color development, they were rinsed with tap water immediately. All the slides were stained with haematoxylin, after which they were observed under a light microscope. Imaging was performed using an optical microscope (BX51, Japan), connected to a camera (DP12, Japan). The positive cell count in 10 fields of 10 sections was obtained using Image J (version 1.48), based on the scale bar set on images, with optical microscope adjusted on the measurement scale."

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