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 | Â | Â | Phospho-Akt (Ser473); (1:25) (Cell Signalling, #3787), 1 h | Â | Â | Â | Â |
Ki67 | Preheated 0.01 M citrate buffer pH 6.0, microwave 800 W, 10 min | Â | 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 | Realâ„¢ (Dako) |
ER | Â | Â | Clone 6F11 (1:40) (Novocastra), 20 min | Â | Â | Â | Â |
PgR | Â | Â | Clone 16 (1:100) (Novocastra), 20 min | Â | Â | Â | Â |
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) | |