Hypoxia-Inducible Factor 1 alpha (HIF-1alpha) Antibody Staining Protocol for Immunohistochemistry


Description: Oxygen availability plays a major role in the regulation of expression of many different genes including erythropoietin, nitric oxide synthase (NOS), heme oxygenase 1 (HO-1), glucose transporters and vascular growth factors necessary for the maintenance of homeostasis in hypoxic conditions. Hypoxia-inducible factor 1 alpha (HIF1 alpha) has been identified as a bHLH-PAS family member that is instrumental in the oxygen-dependent regulation of these genes. HIF1 alpha rapidly accumulates in the nucleus upon exposure to hypoxic conditions where it heterodimerises with the aryl hydrocarbon nuclear receptor translocator ARNT (also referred to as HIF1 beta). Heterodimerisation promotes efficient DNA binding.


Primary Antibody

Name: HIF-1alpha-Carboxyterminal end Antibody

Clone: Rabbit polyclonal

Supplier: Abcam

Catalog Number: ab65979

Dilution: 1:100 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 minutes/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 ºC - 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 in normoxia, nuclear translocation in response to hypoxia
Images: Search image

Additional Information:

Species Reactivity: Human, mouse, rat, rabbit

Fixation: Formalin fixed, paraffin embedded sections

Positive Control: Skin, hypoxia-induced tumors or human placenta

Negative Control: Omit primary antibody, isotype control or 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