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

Name: Cox-2 Antibody

Clone: CX229, Mouse anti-Human

Supplier: Cayman Chemical

Catalog Number: 160112

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.

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