Goat anti rabbit envision system plus hrp

Construction of the TMA and IHC staining was performed as described previously14 (link). Briefly, sections were incubated with 1:100 dilution of anti-KLF14 (21234-1-AP, Proteintech, Wuhan, China), or 1:150 dilution of anti-LDHB (19987-1-AP, Proteintech, Wuhan, China) overnight at 4C, and then incubated with goat anti-rabbit Envision System Plus-HRP (Dako Cytomation) for 30 min at room temperature. After rinsing three times in PBS for 10 min each, the sections were incubated with DAB for 1 min, counterstained with Mayer hematoxylin, dehydrated, and mounted. Phosphate-buffered saline was used as a negative control.
Data were assessed by two independent single-blinded pathologists. A semiquantitative scoring system 14 (link) was used to evaluate both staining intensity (0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining) and the percentage of stained cells (0, <5%; 1, 5%-25%; 2, 26%-50%; 3, 51%-75%; and 4, >75%). The scores for staining intensity and percentage of positive cells were then multiplied to generate the immunoreactivity score for each case15 (link), 16 (link). All cases were sorted into two groups according to the immunoreactivity score. High expression of KLF14 or LDHB was defined as detectable immunoreactions in the nucleus or in cytoplasm with an immunoreactivity score of ≥4.

Construction of the tissue microarray and IHC staining was performed as previously described 14 (link), 15 (link). Briefly, sections were incubated with 1:100 dilution of anti-NOTCH4 (Q99466, Signalway Antibody, Maryland, USA.) overnight at 4C, and then incubated with goat anti-rabbit Envision System Plus-HRP (Dako Cytomation) for 30 min at room temperature. After rinsing three times in PBS for 10 min each, the sections were incubated with DAB for 1 min, counterstained with Mayer hematoxylin, dehydrated, and mounted. Phosphate-buffered saline was used as a negative control.
Data were assessed by two independent single-blinded pathologists. A semiquantitative scoring system 16 (link) was used to evaluate both staining intensity (0, no staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining) and the percentage of stained cells (0, <5%; 1, 5%-25%; 2, 26%-50%; 3, 51%-75%; and 4, >75%). The scores for staining intensity and percentage of positive cells were then multiplied to generate the immunoreactivity score for each case 17 (link), 18 (link). All cases were sorted into two groups according to the immunoreactivity score. High expression of NOTCH4 was defined as detectable immunoreactions in the nucleus with an immunoreactivity score of ≥4.