*2.5. Immunocytochemistry and Toluidine Blue Staining of Heparanase-Treated Chondrocytes*

Chamber slides were brought to room temperate and the PBS replaced with 0.2% Tween 20 for 10 min to permeabilise the cells. After three washes with PBS, the same staining protocol used for immunohistochemistry (see Section 2.2) was followed to reveal the presence of perlecan on the adherent cells, with the addition of a haematoxylin counterstain (diluted 1:3) for 5 s before the slides were mounted in Pertex.

To visualise the presence of glycosaminoglycans, chamber slides were brought to room temperate and the PBS replaced with toluidine blue for 30 s, then washed with distilled water for 5 min. The slides were dehydrated in 70%, 90% and 100% isopropanol (2 min each) and cleared in xylene (2 × 5 min). The slides were mounted in Pertex for imaging.
