Sigma fast 3 30 diaminobenzidine tablet set

Xenograft tumor mass was fixed in 4% w/v paraformaldehyde for 24 h and then included in paraffin. Slide-mounted tissue sections (4 um thick) were deparaffinized in xylene and hydrated serially in 100%, 95%, and 80% ethanol. For immunohistochemistry, endogenous peroxidases were quenched in 3% H₂O₂ in PBS for 1 h, and then, slides were incubated with a primary antibody for 1 h at room temperature (DDX3X, Bethyl laboratories Inc.). Sections were washed three times in PBS, and antibody binding was revealed using the Sigma fast 3,30-diaminobenzidine tablet set (Sigma). The sections were washed, dehydrated, and mounted with resinous mounting medium.

Tissue samples were fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2 and embedded in paraffin. Slide-mounted tissue sections (4-μm thick) were deparaffinized in xylene and serially hydrated in 100%, 95%, and 80% ethanol. Endogenous peroxidases were quenced in 3% H₂O₂ in phosphate-buffered saline (PBS) for 1 h and then slides were incubated with an anti-human primary antibody (10 μg/ml) for 1 h and then with peroxidase-conjugated secondary antibody for 30 min at room temperature (RT). Sections were washed three times in PBS and antibody binding was revealed using the Sigma fast 3,30-diaminobenzidine tablet set (Sigma). Counterstaining was performed using haematoxylin solution. Anti-human MCT1 (H-70), anti-human MCT4 (H-90) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The expression of MCTs was quantified using Remmele scoring system [18 (link)]. The score was calculated by multiplying the number reflecting the dominant stain intensity (0, no detectable stain; 1, weak stain; 2, moderate stain; or 3, strong stain) by the number reflecting the percentage of these positive tumor cells (0, no positive cells; 1, <10%; 2, 10-50%; 3, 51-80%; or 4, >80%). The 12-point scale was categorized in three expression groups: 0 = no expression; 1–5 = weak expression; 6–12 = high expression.