Rabbit anti lgr5

IHC was performed to study the expression of Lgr5 in SCCE tissues, as described previously
[19 (link)]. Briefly, paraffin-embedded tissue sections were baked at 60°C for 2 h, deparaffinized, and rehydrated. After treatment with 3% hydrogen peroxide for 30 min, the sections were put in a high-pressure environment for antigen retrieval. Tissue sections were incubated with rabbit anti-Lgr5 (1:400; Abcam, United states) overnight at 4°C, then treated with anti-rabbit secondary antibody for 40 min, followed by treatment with diaminobenzidine tetrahydrochloride (DAB), and counterstaining with hematoxylin. Human colon cancer tissues with strong Lgr5 staining were used as positive controls, based on previous reports
[20 (link)]. Lgr5 immunostaining was evaluated by two independent observers who were blinded to the clinicopathological characteristics of the patients. Immunostaining scores were awarded by two independent observers according to the percentage and intensity of the stained cells. Positivity values were as follows: 0 (<10%), 1 (10–25%), 2 (25–50%), 3 (50–75%), and 4 (>75%). Intensity values were as follows: 0 (negative), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The final score was calculated by multiplying the above two values. For subsequent analysis, high expression was defined as a final score >4 and low expression was a score ≤4.

Cells were treated with various concentrations of BP compounds for 72 h to detect cancer stem cell markers, as previously described [39 (link)]. Mouse anti-ALDH-1 (Santa Cruz Biotechnology, INC., Dallas, TX, USA), rabbit anti-CD133 (Cell applications, INC., San Diego, CA, USA), mouse anti-CD44, and rabbit anti-β-catenin (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Lgr-5, and rabbit anti-Msi-1 (Abcam, Cambridge, England) were used as primary antibodies for the detection of cancer stemness markers. Rabbit anti-α-tubulin (Cell signaling Technology) was used as an internal standard. All results are representative from at least three independent experiments. Bands were measured by Multi-Gauge 3.0 (Fuji photo film Co., Ltd., Tokyo, Japan) and their relative density calculated based on the density of the α-tubulin bands in each sample. Values were expressed as arbitrary densitometric units corresponding to signal intensity.