On a semiquantitative scale of staining strength the sections varied from weak (11), moderate (5), strong (8) to very strong (8).
A final hybrid score (H) was assigned to each sample, based on the product of a 0 3 scale of staining intensity and of the percentage of positive cells (0 100%), with a possible range of results from 0 to 300.
Staining was graded on a scale of 0 2 (0=negative staining [no staining of any tumor cells], 1=weakly expression [staining of <25% of tumor cells], and 2=high expression [staining of ≥25% of tumor cells]).
For scoring Lgr5 and MMP2 expression, immunohistochemical staining of cells was evaluated according to a score that added a scale of intensity of staining to the proportion of stained cells (Xi et al, 2010).
Cells were scored for intensity of staining on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown).
The intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2(moderate staining, yellowish brown) and 3 (strong staining, brown).
The intensity of staining was recorded on a scale of 0 (no staining), 1 (weak staining, light brown), 2 (moderate staining, yellowish brown) and 3 (strong staining, brown).
The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown).
This scoring system takes into consideration the proportion of positive cells (scored on a scale of 0 2) and staining intensity (scored on a scale of 0 3).
Consistently, α3 staining was observed in the cytoplasm of the tumor cells, as well as in some of the surrounding endothelial cells.After anti-α3 immunostaining was calibrated against background staining obtained with the preimmune serum, all samples were blindly scored for α3 staining intensity on a scale of 0 (no staining) to 3 (high staining).
