Clinical relevance of the reappraisal of negative hormone receptor expression in breast cancer

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Table 2 Technical protocols used by the two IHC methods

Technical steps	IHC methods
	Streptavidin-Biotin-Peroxidase	Peroxidase-Indirect-Polymer
	(90’s)	(currently)
Endogenous peroxidase blocking	2% H₂O₂	3% H₂O₂
Antigenic retrieval	Citrate buffer pH 6.0 in pressure cooker, 6´ in highest pressure	CC1 buffer pH 9.0, 52´
Primary monoclonal antibodies	NCL-ER-6F11 / NCL-PGR (Novocastra), diluted 1:10, 30´, room temperature	ER Ventana 790–4324 (SP1), pre-diluted, 60´, 37°C
Primary monoclonal antibodies		PGR Ventana 760–4296 (1E2), pre-diluted, 28´, 37°C
Secondary antibody	Biotinylated rabbit anti-mouse (E413, Dako), diluted 1:250, 30´	Ultra view universal DAB (760–500, Ventana), 8´, 37°C
Detection systems	StreptABC Complex (K0377, Dako), diluted 1:100, 30´, room temperature	Ultra view universal DAB (760–500, Ventana), 8´, 37°C
Controls	Negative: Primary antibody omission	Breast carcinoma tissue microarray (TMA) including “negative tumour, with normal glandular epithelium, positive tumour with moderate expression (30-70%), and positive tumour with high expression (≈ 100%)”
	Positive: Breast carcinoma positive case