The University of Rochester Center for Musculoskeletal Research has a nice list of protocols for cartilage and bone histology, including for collagen II IHC. That may be a good place to start:

http://www.urmc.rochester.edu/musculoskeletal-research/core-services/histology/protocols.cfm

Dear Mudasir

This article may be useful for you

Effect of collagen type I or type II on chondrogenesis by cultured human articular chondrocytes.

Rutgers M1, Saris DB, Vonk LA, van Rijen MH, Akrum V, Langeveld D, van Boxtel A, Dhert WJ, Creemers LB.

Tissue Eng Part A. 2013 Jan;19(1-2):59-65. doi: 10.1089/ten.TEA.2011.0416. Epub 2012 Sep 13.

Abstract

INTRODUCTION:

Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, however, fibrillar collagens were shown to promote cartilage degradation. The goal of this study was to evaluate the effects of collagen type I and type II coating on chondrogenic properties of in vitro cultured human chondrocytes, and to investigate if collagen-mediated cartilage degradation occurs.

METHODS:

Human chondrocytes of eight healthy cartilage donors were isolated, expanded, and cultured on culture well inserts coated with either collagen type I, type II, or no coating (control). After 28 days of redifferentiation culture, safranin O and immunohistochemical staining for collagen types I, II, X, and Runx2/Cbfa1 were performed and glycosaminoglycan (GAG) and DNA content and release were examined. Further, expression of collagen type I, type II, type X, MMP13, Runx2/Cbfa1, DDR2, α2 and β1 integrin were examined by reverse transcriptase-polymerase chain reaction.

RESULTS:

The matrix, created by chondrocytes grown on collagen type I- and II-coated membranes, resembled cartilage more than when grown on noncoated membranes as reflected by histological scoring. Immunohistochemical staining did not differ between the conditions. GAG content as well as GAG/DNA were higher for collagen type II-coated cartilage constructs than control. GAG release was also higher on collagen type I- and II-coated constructs. Expression of collagen type X was higher of chondrocytes grown on collagen type II compared to controls, but no collagen X protein could be demonstrated by immunohistochemistry. No effects of collagen coating on DDR2 nor MMP-13 gene expression were found. No differences were observed between collagen types I and II.

CONCLUSION:

Chondrocyte culture on collagen type I or II promotes more active matrix production and turnover. No significant differences between collagen types I and II were observed, nor were hypertrophic changes more evident in either condition. The use of collagen type I or II coating for in vitro models, thus, seems a sound basis for in vivo repair procedures

Dear Elias,

Thank you for the response

Actually, I am not able to standardise the procedure of IHC for collagen type II. I have fixed tissues in formalin (buffered)-10%. Kindly if anyone could give some valuable suggestions for staining (IHC).

Thanks

Dear Mudasir, it would be really helpful if you could include some valuable information like:

i) animal (?) or human

ii) Imm-histological approach on previously 10% Formalin = buffered 4% FormAldehyde fixed and later on paraffin-embedded sections

iii) Imm-EM?

regards,

Wolfgang

The University of Rochester Center for Musculoskeletal Research has a nice list of protocols for cartilage and bone histology, including for collagen II IHC. That may be a good place to start:

http://www.urmc.rochester.edu/musculoskeletal-research/core-services/histology/protocols.cfm

Thank you oll for your valuable suggestions...Really wonderful to see scientists from all over coming to rescue

May I add a question? Is there any standardized scoring/protocol to evaluate articular cartilage collagen IHC staining? Thank you

Thank u all