Mouse monoclonal anti cd133 antibody

Formalin-fixed, paraffin-embedded tumor tissue was cut into 4-μm thick sections and immunohistochemistry was performed using a protocol previously reported by our group but with some modifications[15 (link)]. The most representative section of tumor for each case was selected for analysis. We analyzed not only DCLK1 expression, but also the expression of E-cadherin, N-cadherin, vimentin, and Snail as EMT markers, and CD24, CD44, CD133, and epithelial cell adhesion molecule (EpCAM) as CSC markers. The primary antibodies used for immunohistochemistry were: rabbit polyclonal anti-DCLK1 antibody (1:80 dilution; Abcam, Cambridge, MA, United States); rabbit polyclonal anti-Snail antibody (1:80 dilution; Abcam); mouse monoclonal anti-E-cadherin antibody (1:50 dilution; Dako Co., Carpinteria, CA, United States); rabbit monoclonal anti-vimentin antibody (1:100 dilution; Cell Signaling, Danvers, MA, United States); rabbit polyclonal anti-N-cadherin antibody (1:100 dilution; Abcam); goat polyclonal anti-CD24 antibody (1:20 dilution; Santa Cruz Biotechnology, Dallas, TX, United States); mouse monoclonal anti-CD44 antibody (1:50 dilution; Dako Co); mouse monoclonal anti-CD133 antibody (1:10 dilution; Miltenyi Biotec, Gladbach, Germany); and mouse monoclonal anti-EpCAM antibody (1:500 dilution; Cell Signaling).

Cervical cancer specimens of all patients, which were fixed in formalin and embedded in paraffin (FFEP), were collected to assess the expressions of ITGA7, CD133, and ALDH1 by IHC assay. The mouse monoclonal anti‐ITGA7 antibody (1:50 dilution, Santa Cruz Biotechnology), the mouse monoclonal anti‐CD133 antibody (1:50 dilution, Miltenyi biotec), and the mouse monoclonal anti‐ALDH1 antibody (1:100 dilution, Abcam; Cambridge, UK) were applied as primary antibody. The goat anti‐mouse IgG (H&L) (Abcam) with 1:2000 dilution was used as secondary antibody.¹³, ²⁰, ²¹ After staining, the protein expression was calculated using a semiquantitative scoring method. Briefly, in every slide of specimens, five selected high‐power fields (HPF, ×400) were independently counted by two pathologists without prior knowledge of the clinicopathological information to evaluate the intensity and density of cells. Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), while staining density was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The product of staining intensity score and staining density score was the final IHC score, which was classified as low expression (final IHC score ≤3) and high expression (final IHC score >3).