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Table 1 Details of the immunohistochemical procedure used for each marker*

From: Extreme loss of immunoreactive p-Akt and p-Erk1/2 during routine fixation of primary breast cancer

IHC procedure Antigen Retrieval Endogenous Peroxidase Blocking Monoclonal Antibody Secondary Antibody Signal Amplification Signal Detection Kit used
p-Erk1/2 Preheated target retrieval solution (1×), water bath 97 ± 2°C, 30 min Phosphate buffer containing hydrogen peroxide, 5 min Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204); (1:100) (Cell Signalling, #4376), 1 h Rabbit linker, 15 min Polymer conjugated to horseradish peroxidase (HRP) and goat antirabbit and antimouse immunoglobulin, 30 min   EnVision™ FLEX+ (Dako)
p-Akt    Phospho-Akt (Ser473); (1:25) (Cell Signalling, #3787), 1 h     
Ki67 Preheated 0.01 M citrate buffer pH 6.0, microwave 800 W, 10 min   Clone MIB-1 (1:40) (Dako), 20 min Biotinylated goat antimouse and antirabbit immunoglobulins, 15 min Streptavidin conjugated to HRP, 15 min DAB (chromogen) in hydrogen peroxide (substrate), 10 min Real™ (Dako)
ER    Clone 6F11 (1:40) (Novocastra), 20 min     
PgR    Clone 16 (1:100) (Novocastra), 20 min     
HER2 Preheated 0.01 M citrate buffer, water bath 97 ± 2°C, 40 min 3% hydrogen peroxide, 5 min Rabbit antihuman HER2 protein, 30 min Polymer conjugated to HRP and goat antirabbit immunoglobulin, 30 min   HercepTest™ (Dako)
  1. *ER, oestrogen receptor: PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; HRP, horseradish peroxidase, DAB, 3,3'-diaminobenzidine.