Table 2

Characteristics for anti-HER and anti-Ki-67 immunostaining and scoring
Characteristic	HER1	HER2	HER3	HER4	Ki-67

Antibody	Mouse mAb	Rabbit pAb	Mouse mAb	Rabbit mAb	Mouse mAb
Origin	Dako	Dako	NanoTools	Cell Signaling	Dako
Clone	2–18C9	-	5A12	83B10	MIB-1
Concentration of primary antibody	EGFR pharmDX	HercepTest (2.5 μg/ml)	5 μg/ml culture supernatant	0.7 μg/ml	1.6 μg/ml
Staining pattern	Membrane	Membrane	Cytoplasm and membrane	Cytoplasm and membrane	Nucleus
Epitope retrieval	Proteinase K	Heat induced, 10 mM citric acid buffer, pH 6.0	Heat induced, 10 mM citric acid buffer, pH 7.3	Heat induced, 10 mM sodium-citric acid pH 6.0	Heat induced, 10 mM citric acid buffer, pH 7.2
Blocking	Endogenous peroxidase blocking	Endogenous peroxidase blocking	Endogenous peroxidase blocking	Endogenous peroxidase blocking	Endogenous peroxidase blocking
Primary antibody	Overnight, 4°C	Overnight, 4°C	Overnight, 4°C	Overnight, 4°C	30 min, room temperature
Detection system	EnVision™ Dual Link System (Dako); DAB + chromogenic substrate	EnVision™ Dual Link System (Dako); DAB + chromogenic substrate	EnVision™ Dual Link System (Dako); DAB + chromogenic substrate	EnVision™ Dual Link System (Dako); DAB + chromogenic substrate	iVIEW™ DAB Detection Kit (Ventana)
Scoring in accordance with	EGFR pharmDx guidelines	HercepTest guidelines	EGFR pharmDx guidelines	EGFR pharmDx guidelines	Manufacturer's guidelines

mAb, monoclonal antibody; pAb, polyclonal antibody; DAB, diaminobenzidine; EGFR, epidermal growth factor receptor.
Sassen et al. Breast Cancer Research 2008 10:R2 doi:10.1186/bcr1843