Heme Oxygenase-1 (HO-1) Antibody Staining Protocol for Immunohistochemistry


Description: Heme oxygenase-1 (HO-1) or HSP32 is the inducible isoform of heme oxygenase which catalyzes the NADPH, O2 and cytochrome P450 reductase dependent oxidation of heme to carbon monoxide, iron and billiverdin that is immediately reduced to bilirubin. These products of the HO reaction have important physiological effects: carbon monoxide is a potent vasodilator; biliverdin and its product bilirubin are potent antioxidants; “free” iron increases oxidative stress and regulates the expression of many mRNAs by affecting the conformation of iron regulatory protein (IRP)-1 and its binding to iron regulatory elements (IREs) in the 5’- or 3’ –UTRs of the mRNAs. To date, three heme oxygenase isoforms HO-1, HO-2, and HO-3 have been identified. HO-1 or HSP32, a major heat shock/stress response protein is ubiquitous and its mRNA as well as its activity can be increased several-fold by heme, other metalloporphyrins, transition metals and stimuli that induce cellular stress. HO-1 expression has been shown to increase in benign prostatic hyperplasia (BPH) and malignant prostate tissue suggesting a role for stress protein in the pathogenesis of BPH and prostate cancer. There is  recent data which indicates the ability of peroxynitrite (ONOO-) to modulate the expression of HO-1 and suggests that the heme oxygenase pathway contributes to protection against the cytotoxic action of ONOO- which is a potent oxidizing agent generated by the interaction of nitric oxide (NO) and the superoxide anion. ONOO- rapidly decomposes to a highly reactive hydroxyl radical and nitrogen dioxide, both of which cause oxidative damage.


Primary Antibody

Name: HO-1 Antibody

Clone: Rabbit polyclonal

Supplier: StressGen Biotechnologies

Catalog Number: OSA-150C

Dilution: 1:5000 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/nuclear
Images: Search image

Additional Information:

Tissue Type: Prostate cancer

Fixation: Formalin fixed paraffin sections

Positive Control: Prostate cancer

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


Not tested on frozen sections.