Carbonic Anhydrase IX (CA IX) Antibody Staining Protocol for Immunohistochemistry


Description: Carbonic anhydrase (CA) is an enzyme that assists rapid interconversion of carbon dioxide and water into carbonic acid, protons, and bicarbonate ions. It is abundant in all mammalian tissues. Because of its functionality, it has become an important diagnostic marker for various cancers, most notably renal cell carcinoma (RCC). There are many genes that are inducible by hypoxia, via HIF-1 alpha. CA IX is one of the most inducible genes because of its stability and location within the membrane. Carbonic anhydrases have a widespread role in regulating pH in normal tissues, by regulating hydrogen ion (H+) flux. The pH is important in cell death under hypoxia, thus a blockade of CA IX results in increased cell death under hypoxia. Therefore, CA IX has become a reliable histochemical marker of hypoxia.


Primary Antibody

Name: Carbonic Anhydrase IX (CA IX) Antibody

Clone: Rabbit anti-Human

Supplier: Novus Biologicals

Catalog Number: NB100-417

Dilution: 1:2000 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: 60min/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: Renal carcinoma
Fixation: Formalin-fixed paraffin sections
Positive Control: Renal carcinoma
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