Leica dm3000 led

Immunohistochemical analyses were done in 4 μm femur cartilage samples. Antigen retrieval was performed by incubation with 0.1% trypsin from bovine pancreas (Sigma-Aldrich, USA) in 0.1% CaCl₂. Inflammatory markers were visualized using anti-metalloproteinase (MMP)-13 (R&D systems, USA, MAB511, 30 μg/ml) and anti-ciclooxigenase (COX)-2 (sc-1745; Santa Cruz Biotechnology, USA, 1/100 dilution) antibodies as previously described (30 (link)). A secondary biotinylated anti-mouse and anti-goat IgG was used respectively for detection of positive signal through a horseradish peroxidase linked to an avidin/biotin complex (ABC) (Vector Laboratories, USA) using 3,3 diaminobenzidine tetra-hydrochloride as chromogen (Dako, Denmark). Sections were counterstained with Haematoxylin, dehydrated and mounted in DPX (Merck Millipore, USA). Positive immunoreactivity was evaluated in x20 magnification photographs obtained using a Leica DM3000 LED digital micro-imaging instrument (Leica Microsystems, USA). Each image was analyzed using the Image J software (National Institutes of Health, USA) and the percentage of positive area was calculated as previously described (30 (link), 31 (link)).

The analysis of the GP chondrocyte column organization was performed in the knee sections obtained eight weeks after DMM surgery from operated and non-operated animals. 4 µm sagittal paraffin-embedded knee sections were stained with Haematoxylin–Eosin. Joint sections were photographed with a Leica DM3000 LED digital micro-imaging instrument (Leica, Microsystems, Inc. Buffalo Grove, IL, USA) at the central region of the tibia at × 40 magnification, and chondrocyte histomorphometry was evaluated as described elsewhere^{27 (link),46 (link)}. Briefly, a chondrocyte column was defined as a chondron containing a minimum of three chondrocytes, located no more than 20 pixels apart one from each other, and with angles between them ranging from -155° to -179° or 155° to 180°. These parameters were computed as previously described by Killion et al^{27 (link)}. Once the columns were established in each sample, the number of cells was quantified using the Image J Software (NIH, USA) cell counter plug-in. Then, the percentage of cells in columns was obtained by calculating the ratio ‘cells in columns’/ ‘total cells in the GP’. Column Index (CI) was calculated multiplying the percentage of cells in columns by their respective length^{27 (link)}.