MUC-1 Antibody Staining Protocol for Immunohistochemistry


Description: Muc1 is a heavily O-glycosylated transmembrane protein expressed on most secretory epithelium, including mammary gland and some hematopoietic cells. It is expressed abundantly in lactating mammary glands and overexpressed in >90% breast carcinomas and metastases. In normal mammary gland it is expressed in apical surface of glandular epithelium. In breast cancer Muc1 is overexpressed; is underglycosylated and the apical localization is lost. Muc1 is transcribed as a larger precursor which is cleaved to form a larger mucin like subunit (265-400kD) and a smaller subunit (14-28kD) noncovalently associated with each other. Transgenic Muc1 has been shown to associate with all four erbB receptors and localize with erbB1 (EGFR) in lactating glands1. Muc1 can act as a ligand for ICAM-1 on HUVEC cells; it can bind beta-catenin, GSK3beta and it associates with Grb2-SOS upon phosphorylation. Epithelial Membrane Antigen, also referred to as EMA, CA15-3, Polymorphic Epithelial Mucin, PEM, Sialomucin or Episialin and MUC-1 is a glycoprotein of variable molecular weight, expressed by many glandular epithelial cells. Epithelia of breast, (acini and ducts), exocrine pancreas, bladder, gastrointestinal tracts, thyroid, endometrium and respiratory tract are generally EMA positive.


Primary Antibody

Name: MUC-1 Antibody

Clone: 0.N.272, Mouse anti-Human

Supplier: US Biological

Catalog Number: E3414-19X

Dilution: 1:200 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 min/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: Cytoplasmic/cell membrane
Images: Search image

Additional Information:
Tissue Type: Breast or colon carcinoma. MCF-7 and T47D cells
Fixation: Formalin fixed paraffin sections
Positive Control: Breast or colon carcinoma. MCF-7 and T47D cells
Negative Control: Omit primary antibody, isotype control, 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