Matrix changes in affected proteoglycan-induced spondylitis (PGISp) mouse vertebrae. (A) Toluidine blue staining in unaffected week-12 control vertebrae shows clear staining in the vertebral growth plate cartilage and also in the nucleus pulposus (×40). Note the lack of staining in the bone. (B) Further tissue delineation is shown using Gruber's intervertebral disc (IVD) stain (×40). Collagen in the bone and annulus fibrosus is stained red, and proteoglycan (PG) in the cartilage and nucleus pulposus is stained blue. The cellular nature of the IVD is clearly visible. Toluidine blue staining of the same affected sacral joint shown in Figure 1 shows that the majority of the excess matrix laid down is PG-rich. However, note the fringes of this matrix stain negative for PG (red arrowheads); ×40 (C), ×100 (D). Gruber's IVD stain further illustrates the PG-rich nature of the excess matrix; ×40 (E), ×100 (boxed area in F). However the PG-negative fringes also stain positive for collagen (black arrowheads).