 |
|
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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
Not tested on frozen sections. |
References: