Diaminobenzidine tetrahydroxychloride solution dab

STAT3 and p-STAT3 (S727) were detected in the paraffin-embedded CCA tissues using a standard immunohistochemistry protocol. Briefly, the tissues were incubated with 1: 100 of anti-STAT3 or anti-p-STAT3 (S727) at room temperature, overnight. The Envision-Plus HRP system (Dako, Carpinteria, CA) against the primary antibody was applied at room temperature for 2 h. The peroxidase activity was observed using diaminobenzidine tetrahydroxychloride solution (DAB; Dako) as a substrate, and counter stained with hematoxylin. The frequency of target molecules were semi-quantitatively scored on the basis of percentage of the positively stained cells as 0 = negative; 1–25% = 1; 26–50% = 2; and >50% = 3. The intensity of stained cells was scored as weak = 1, moderate = 2 and strong = 3. The immunohistochemistry (IHC) index was calculated as frequency × intensity.

The lectin histochemistry was performed as previously described^{11 (link)}. Briefly, non-specific background was blocked with 0.5% periodate-treated BSA (tBSA) for 30 min at room temperature. The tissue sections were then incubated with 1:100 biotinylated UEA-I (Vector laboratories, Burlingame, CA) at room temperature for 2 h and with 1:100 streptavidin-conjugated HRP (Invitrogen, Camarillo, CA) for 40 min. The peroxidase activity was developed by diaminobenzidine tetrahydroxychloride solution (DAB; Dako, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin (Bio-optica; Milan, Italy). The TFG of each tissue section was scored under light microscopy according to the Allred scoring system^{50 (link)}. In brief, the percentage of positive stained cells were graded as 0 = negative, 1–25% = 1, 26–50% = 2, 51–75% = 3 and >76% = 4. The intensities of the stained cells were scored as 1 = weak, 2 = moderate, 3 = strong. The TFG score was calculated as frequency x intensity and classified into low and high according to the medians of the TFG scores.