CD44 Antibody Staining Protocol for Immunohistochemistry


Description: CD44 is a type 1 transmembrane glycoprotein also known as Phagocytic Glycoprotein-1 (pgp-1) and HCAM. CD44 is the receptor for hyaluronate and exists as a large number of different isoforms due to alternative RNA splicing. The major isoform expressed on lymphocytes, myeloid cells, and erythrocytes is a glycosylated type 1 transmembrane protein. Other isoforms contain glycosaminoglycans and are expressed on hematopoietic and non-hematopoietic cells. CD44 is involved in adhesion of leukocytes to endothelial cells, stromal cells, and the extracellular matrix.


Primary Antibody

Name: CD44 Antibody

Clone: MAC329, Rat anti-Pig

Supplier: Research Diagnostics

Catalog Number: RDI-PGCD44abrt

Dilution: 1:20 - 1:40 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: Cell membrane
Images: Search image

Additional Information:
Species Reactivity: Porcine
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Tonsil, spleen, cardiac tissues
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


Not need to perform antigen retrieval on acetone fixed frozen sections