c-erbB-2/HER-2/neu (phospho-specific) Antibody Staining Protocol for Immunohistochemistry


Description: c-erbB-2 (HER-2/neu) is a 185 kDa receptor tyrosine kinase belonging to the epidermal growth factor receptor family, which also includes EGFR (HER-1), c-erbB-3 (HER-3), and c-erbB-4 (HER-4). Amplification of the HER-2 gene with overexpression of the protein occurs in 20-30% of breast cancers, and tumor progression in mammary cancer is associated with elevated levels of tyrosine-phosphorylated neu and erbB-3. Tyr is the phosphorylation site that is most tightly linked to oncogenic transformation and coupling to the ras/MAP kinase signaling pathway. Cases of invasive breast carcinoma with the phosphorylated receptor have displayed aggressive clinicopathological features and adverse prognoses. Activation of HER-2 is frequent in ductal carcinoma in situ suggesting that HER-2 signaling plays a critical role in the early stages of breast tumorigenesis.


Primary Antibody

Name: c-erbB-2/HER-2/neu (phospho-specific) Ab-18 Antibody

Clone: PN2A, Mouse

Supplier: Lab Vision

Catalog Number: MS-1072-P

Dilution: 1:20 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 minutes/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Membrane
Images: Search image

Additional Information:
Tissue Type: Breast
Fixation: Formalin fixed paraffin sections
Positive Control: EGF-treated SKBR3 cells. About 5% of breast carcinomas are positive for phospho-c-erbB-2/HER-2/neu oncoprotein.
Negative Control: Omit primary antibody, isotype control or absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary