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iNOS Antibody Staining Protocol for Immunohistochemistry
Description: Nitric oxide (NO) is an inorganic, gaseous free radical that carries a variety of messages between cells. Vasorelaxation, neurotransmission and cytotoxicity can all be potentiated through cellular response to NO. NO production is mediated by members of the nitric oxide synthase (NOS) family. NOS catalyzes the oxidization of L-arginine to produce L-citrulline and NO. Two constitutive isoforms, brain or neuronal NOS (b or nNOS, type I) & endothelial cell NOS (eNOS, type III), and one inducible isoform (iNOS, type II), have been cloned. All NOS isoforms contain calmodulin, nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) binding domains. Nitric oxide synthase is expressed in liver, macrophages, hepatocytes, synoviocytes, stimulated glial cells and smooth muscle cells. Cytokines such as interferon-gamma (IFN), tumor necrosis factor (TNF), interleukin-1 and -2, and lipopolysaccarides (LPS) cause an increase in iNOS mRNA, protein, and activity levels. Protein kinase C-stimulating agents exhibit the same effect on iNOS activity. After cytokine induction, iNOS exhibits a delayed activity response which is then followed by a significant increase in NO production over a long period of time. Human iNOS is regulated by calcium/calmodulin (in contrast with mouse NOS2).
Primary Antibody
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Name: iNOS Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Abcam |
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Catalog Number: ab15323 |
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Dilution: 1:100 - 1:200 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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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/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/extracellular matrix |
| Images: Search image |
Additional Information:
| Species reactivity: Human, mouse, rat |
| Fixation: Formalin fixed paraffin sections or acetone fixed frozen sections |
| Positive Control: Lung, human tonsil or skin |
| 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:
Dilution depends on detection system |
References: