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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
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Name: CD44 Antibody |
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Clone: MAC329, Rat anti-Pig |
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Supplier: Research Diagnostics |
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Catalog Number: RDI-PGCD44abrt |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
Not need to perform antigen retrieval on acetone fixed frozen sections |
References: