Anti collagen 2 clone 2 4c11

Sections were washed followed by a 15 min digestion with 0.1% pepsin at pH 3.5 for a facilitated antibody access to the target epitopes. Type I and II collagen were immunolocalized by the immunoperoxidase ABC technique (Vector, Burlingame, CA, USA). As primary antibodies anti-collagen II (clone II-4C11; Calbiochem Merck, Schwalbach, Germany) and monoclonal CD31 mouse anti-rabbit antibodies (clone JC-70A IgG1 light chain type kappa; Abcam, Cambridge, UK) were used. After staining with biotin conjugated polyclonal goat anti mouse IgG secondary antibody (Jackson, West Grove, PA, USA), positive signals were visualized by nickel- and cobalt-enhanced 3,3′-diaminobenzidine (DAB). Two blinded scorers analyzed the sections semi-quantitatively according to the proposed scoring system. Percentage of content of collagen I or collagen II was determined by comparing DMMB stained slides with immunohistochemically stained slides.

For immunohistochemical analysis, frozen sections were prepared as previously described [6 (link),12 (link),21 (link)]. Embedded in Tissue-Tek OCT (Sakura Finetek, Tokyo, Japan), the samples were washed in phosphate-buffered saline and subsequently digested with 0.1 % pepsin at pH 3.5 for 15 min to facilitate antibody access to the target epitopes. Type II collagen was immunolocalized by the immunoperoxidase ABC technique (Vector, Burlingame, CA, USA). Anti collagen II (clone II-4C11; Calbiochem Merck, Schwalbach Germany) was used as primary antibody with an antibody dilution of 1:100. After staining with biotin-conjugated polyclonal goat anti-mouse IgG secondary antibody (Jackson, West Grove, PA, USA), positive signals were visualized using nickel- and cobalt-enhanced 3,3’-diaminobenzidine (DAB).

The frozen sections, embedded in Tissue-Tek OCT (Sakura Finetek, Tokyo, Japan), were washed in a phosphate-buffered saline and digested with 0.1% pepsin at pH 3.5 for 15 minutes to facilitate antibody access to the target epitopes. Type II collagen was immunolocalized by the immunoperoxidase ABC technique (Vector, Burlingame, CA, USA). As primary antibodies, anticollagen II (clone II-4C11; Calbiochem Merck, Schwalbach Germany) was used. The antibody dilution was 1 : 100. After staining with biotin conjugated polyclonal goat anti-mouse IgG secondary antibody (Jackson, West Grove, PA, USA), positive signals were visualized by nickel and cobalt-enhanced 3,3′-diaminobenzidine (DAB) [13 (link), 23 (link)].
Concerning the evaluation of the content of collagen type II, the percentage of collagen type II stained area within the extracellular matrix in the porous area of the scaffolds was assessed in comparison to the extracellular matrix in the DMMB-stained samples.