Cox-2 Antibody Staining Protocol for Immunohistochemistry


Description: Prostaglandins are a diverse group of autocrine and paracrine hormones that mediate many cellular and physiologic processes. Prostaglandin H2 (PGH2) is an intermediate in formation of the prostaglandins. Two prostaglandin synthases that catalyze the formation of PGH2 from arachidonic acid (AA) are cyclooxygenase-1 and cyclooxygenase-2. Cyclooxygenase-2, or Cox-2, is efficiently induced in migratory cells responding to pro-inflammatory stimuli and is considered to be an important mediator of inflammation. An alternative form of the protein, designated Cox-1, is constitutively expressed in most tissues and is thought to serve in general “housekeeping” functions. Both enzymes are targets for the nonsteroidal therapeutic anti-inflammatory drugs, NSAIDs. Cox-2 migrates with an apparent molecular weight of between 72-74 kDa, while Cox-1 is somewhat smaller, migrating at 70 kDa.


Primary Antibody

Name: Cox-2 (M-19) Antibody

Clone: Goat polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-1747

Dilution: 1:100 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: Membrane/cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Colon, kidney
Fixation: Formalin fixed paraffin sections
Positive Control: Colon, kidney
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