MUC-4 Antibody Staining Protocol for Immunohistochemistry


Description: Mucins are large, highly O-glycosylated proteins that can be classified into two main groups: secreted mucins which include gel-forming (MUC2, MUC5AC, MUC5B, and MUC6) and non-gel-forming (MUC7), and membrane-bound mucins (MUC1, MUC3, MUC4, and MUC12). MUC4 has been found to be a heterodimeric glycoprotein complex that consists of ASGP-1 (ascites sialoglycoprotein-1), an O-glycosylated mucin subunit, and ASGP-2, an N-glycosylated transmembrane subunit. Sialomucin complex (SMC, rat homologue of human MUC4) expression is found in normal secretory epithelial tissues in the adult rat, where the protein has multiple regulatory mechanisms(1). SMC is found in normal virgin mammary gland, elevated in the lactating gland, and further amplified in rat mammary adenocarcinoma. Transforming growth factor-beta (TGF-β) inhibits SMC expression. Abnormal expression of MUC4/SMC is found in several human epithelial tumors such as, breast, lung, cervix, and pancreas carcinomas. Unregulated MUC4 may promote tumor metastasis, which suggests that it has an important role in human tumor biology. For example, several studies indicate that the cytoplasmic tail of the transmembrane mucin is involved in cell-signalling events such as Sos-ras pathway, beta-catenin, and ErbB2 signalling pathway.


Primary Antibody

Name: MUC-4 Antibody

Clone: 1G8, Mouse anti-Rat

Supplier: Zymed Labs

Catalog Number: 35-4900

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: Rat mammary adenocarcinoma, breast, colon, pancreas carcinomas.
Fixation: Formalin fixed paraffin sections
Positive Control: Rat mammary adenocarcinoma, breast, colon, pancreas carcinomas.
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