HIF-1-alpha Antibody Staining Protocol for Immunohistochemistry

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

Description: HIF-1α Hypoxia contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease. HIF-1 is a nuclear protein that activates gene transcription in response to reduced cellular O2 concentration. HIF-1 activates the transcription of EPO, VEGF, iNOS, heme oxygenase-1 and several other critical intracellular responses to hypoxia. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits. Both subunits are induced by hypoxia and rapidly decay upon return to normoxia. Recent research suggests that ability to regulate hypoxia-inducible factors may be related to tumor-related angiogenesis in certain cancers.

Primary Antibody

Name: HIF-1-alpha Antibody

Clone: ESEE122, Mouse monoclonal

Supplier: Novus Biologicals

Catalog Number: NB100-131

Dilution: 1:1,000 - 1:10,000 depending on detection system using IHC-Tek^TM 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-Tek^TM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-Tek^TM Epitope Retrieval Solution (Cat# IW-1100)

Heat/Cool Temperature: 95 ºC - 100 ºC/room temperature

Heat/Cool Time: 45 minutes/20 minutes

Detection Methods

Standard Method: ABC Method or LSAB Method

Enhanced Method: Polymeric Methods - Recommended methods for this HIF-1-alpha antibody

Chromogen Substrate

Reagent: DAB

Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Cytoplasmic in normoxia, nuclear translocation in response to hypoxia

Images: Search image

Additional Information:

Species Reactivity: Mouse, rat, human, cow

Fixation: Zinc fixed or Formalin fixed paraffin sections

Positive Control: Mouse or human 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

Notes:

1. Not tested on frozen sections.

2. Antigen retrieval with EDTA/Tris-EDTA produces high background staining and is NOT recommended for HIF-1 alpha staining.

3. When Clone ESEE122 used for mouse tissue, mouse on mouse blocking method is recommended to reduce unspecific background staining.

4. Standard ABC or LSAB method is NOT sensitive enough for this antibody so more sensitive detection systems are recommended such as ImmPRESS (Vector Labs) or Envision System (Dako).

5. Formalin fixed tissues tend to produce weaker staining so Zinc fixative is recommended for this antibody.

References:

Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, Buechler P, Isaacs WB, Semenza GL, Simons JW. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res. 1999 Nov 15;59(22):5830-5.