Collagen Type II Antibody Staining Protocol for Immunohistochemistry


Description: In Western blotting (but not ELISA), MAB8887 recognizes both a1(II) and a3(XI) chains which have identical primary structure. In Western blotting, MAB8887 reacts with the TCA fragment of lathyritic type II collagen after digestion with mammalian collagenase. It also reacts with pepsin-digested type II collagen. It will not react with intact Collagen II under traditional conditions. Its epitope is localized in the triple helix of type II collagen. It shows no cross-reaction with type I or type III collagen. Immunoblotting of CNBr peptides of collagen II shows that MAB8887 reacts with CB11 (25kDa) fragment which is the site of immunogenic and arthritogenic epitopes along the intact type II molecule.


Primary Antibody

Name: Collagen Type II Antibody

Clone: 6B3, Mouse anti-Chicken

Supplier: Chemicon

Catalog Number: MAB8887

Dilution: 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.

Incubation Time/Temp: 60 minutes/room temperature

Antigen Retrieval
Device: Incubator
Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature: 37ºC/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: Collagen fibers
Images: Search image

Additional Information:
Tissue Type: Cartilage
Fixation: Formalin fixed paraffin sections
Positive Control: Cartilage
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



1. Cultured Autologous Chondrocyte Implantation for Cartilage Repair by LEESA M. BARONE, PhD, Genzyme Tissue Repair, Cambridge, Massachusetts.

2. Collagen Type II Immunohistochemistry Articles on PubMed