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Cox-2 Antibody Staining Protocol for Immunohistochemistry
Description: COX catalyzes the first step in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins: the conversion of arachidonic acid to prostaglandin H2. Recent discoveries of the induction of COX by a variety of stimuli such as phorbol esters, lipopolysaccharides, and cytokines led to the hypothesis that the inducible form of COX, COX-2, is responsible for the biosynthesis of prostaglandins under acute inflammatory conditions. COX-2 is a 72 kDa protein which has been cloned from a variety of species including human, mouse, rat and sheep.
Primary Antibody
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Name: Cox-2 Antibody |
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Clone: CX229, Mouse anti-Human |
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Supplier: Cayman Chemical |
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Catalog Number: 160112 |
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Dilution: 1:250 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 - 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 |
| Images: Search image |
Additional Information:
| Tissue Type: Brain (hippocampus, piriform cortex), colon |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Brain (hippocampus, piriform cortex), colon |
| 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:
1. Yamashita H, Osaki M, Ardyanto TD, Osaki M, Yoshida H, Ito H (2004) Cyclooxygenase-2 in human malignant fibrous histiocytoma: correlations with intratumoral microvessel density, expression of vascular endothelial growth factor and thymidine phosphorylase. Int J Mol Med. 14(4):565-70. PubMed Abstract