Abc elite pk 6100

Tissues were collected and fixed in fixative solution containing 270 μl of formaldehyde and 80 μl of gluteraldehyde in 10 ml PBS for 30 min at room temperature. The tissues were washed three times in PBS, and then either incubated with X-gal overnight for whole-mount staining, or embedded in was optimal cutting temperature compound (OCT), sectioned, fixed a second time with fixative solution for 10 min and incubated overnight with X-gal. For immunohistochemistry, slides were fixed in acetone at −20 °C for 2 min, rinsed three times in PBS, and incubated in Dako Ready-to-use Protein Block (Dako Cat. No. X0909) for 10 min at room temperature. Slides were then incubated with primary antibody (polyclonal anti-human VWF, Dako Cat. No. A0082) 1:100 at 4 °C overnight. The slides were then rinsed three times in PBS, blocked with peroxidase (1 ml H₂O₂ in 150 ml methanol) for 10 min at room temperature and washed again before incubating with secondary antibody (biotinylated goat anti-rabbit, Dako Cat. No. E0432). After washing in PBS, the slides were incubated with tertiary antibody (Vector Labs ABC Elite PK-6100) and then processed for AEC staining. Finally, the slides were counterstained with Hematoxylin and saturated Lithium Carbonate Solution and sealed with cover slips in aqueous mounting medium.

Tissue samples were evaluated for indirect immunostaining with the mouse monoclonal antibodies Col2A1 (M2139) and MMP-13 (C-3) from Santa Cruz Biotechnology, using a biotinylated anti-mouse secondary antibody (Donkey anti-mouse IgG (H + L) Biotin, Millipore, Burlington, MA, USA) and the avidin–biotin complex (ABC Elite PK-6100, Vector, Burlingame, CA, USA). Antibody binding was revealed with the DAB-peroxidase kit (Peroxidase Substrate Kit DAB SK-4100 Vector) and counterstaining with Crystal Violet (C5042, Sigma, St. Louis, MO, USA).
The presence of type II collagen (Col2A1) is related to the presence of healthy tissue, which, when hypertrophied after damage, begins to be replaced by type X collagen. On the other hand, metalloproteinase 13 (MMP- 13) is the most widely used marker of inflammation in the evaluation of cartilage, as it is the main protease in articular cartilage degradation [21 ,22 (link)].