Modified Periodic Acid-Schiff (PAS) is an alternative to Safranin O for discriminating bone-cartilage interfaces

Safranin O is a cationic dye that binds to proteoglycans in cartilage and is routinely used to assess growth plate dynamics and/or fracture repair at bone-cartilage interfaces. When used with a counterstain such as fast green, Safranin O can offer exquisite differentiation of cartilage from surrounding bone. However, various decalcification and processing methods can deplete proteoglycans, rendering inconsistent, weak, or absent Safranin O staining with indiscriminate bone-cartilage boundaries. We sought to develop an alternative staining method that preserves the contrast of bone and cartilage in cases of proteoglycan depletion. Periodic acid-Schiff (PAS) is a carbohydrate-specific stain used to demonstrate glycogen and other polysaccharides, such as the mucopolysaccharides in cartilage. This two-step reaction involves exposure to periodic acid, which oxidizes glycol groups to aldehydes, followed by Schiff’s reagent, which turns the aldehydes a rose-violet color. PAS preferentially targets glycol groups with a neutral charge, making this reaction more sensitive than Safranin O for staining cartilage matrix. We developed a simple and novel modification of a standard PAS protocol using Weigert’s iron hematoxylin and light green stains that produces a contrast between bone (blue-green) and cartilage (purple).Juvenile mouse fore- and hindlimb bones (N=87) were fixed in formalin, decalcified in 10% EDTA and processed for routine paraffin histology. Modified PAS staining was performed using a commercially available kit (Sigma-Aldrich) with several modifications to the manufacturer’s protocol: sections were exposed to periodic acid for 5 minutes, rinsed, immersed in Schiff’s reagent for 10 minutes, rinsed and counterstained with Weigert’s iron hematoxylin, after which they were “blued” in PBS, rinsed, and stained with 1% light green.In all samples tested, this modified PAS method consistently rendered purple staining in cartilage and blue-green staining in bone. Importantly, our technique provided a clear distinction of the bone-cartilage interface even when Safranin O staining did not (Figure 1). This modified PAS protocol provides a practical solution for differentiating bone and cartilage when Safranin O staining is not detected following decalcification and paraffin processing. This method can be a useful alternative for studies in which identification of the bone-cartilage interface is essential but may not be preserved with standard staining approaches.