*3.5. Perlecan and Toluidine Blue Staining Is More Prominent in Chondrocyte Clusters*

There was immunostaining for perlecan in some cultured cells, some apparently in the cytoplasm and also associated with the cell membrane. This appeared strongest when cells were in clusters, which were more common in cultures without exposure to heparanase (Figure 8A).

Metachromasia with toluidine blue staining for glycosaminoglycans was mostly weak with no consistent difference in pattern between control and heparanase conditions (Figure 8B). However cell, clusters, where present, tended to have stronger toluidine blue staining.

**Figure 8.** Immunocytochemistry and toluidine blue staining of heparanase-treated chondrocytes (two representative donor examples shown). (**A**) Chondrocytes cultured in chamber slides were treated with heparanase for 48 h and stained for perlecan (*n* = 4). Chondrocytes in untreated controls tended to remain in clusters that stained more intensely for perlecan compared to heparinase-treated chondrocytes. (**B**) Toluidine blue staining revealed no noticeable trend. However, cell clusters in both controls and heparanase conditions had stronger staining than individual cells. Scale bars = 50 µm.
