Modified Periodic Acid‐Schiff (PAS) Is an Alternative to Safranin O for Discriminating Bone–Cartilage Interfaces

Cartilage histomorphometry is often performed on decalcified, paraffin‐embedded bone sections, which provide versatility in staining applications from basic morphology to immunohistochemistry. 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 methodology that preserves the contrast of bone and cartilage in cases of proteoglycan depletion that can be applied when other cartilage stains are unsuccessful. Here, we describe and validate a modified periodic acid‐Schiff (PAS) protocol that we developed using Weigert's iron hematoxylin and light green stains as an alternative to safranin O for discriminating bone–cartilage interfaces of skeletal tissues. This method provides a practical solution for differentiating bone and cartilage when safranin O staining is not detected after decalcification and paraffin processing. The modified PAS protocol can be useful for studies in which identification of the bone–cartilage interface is essential but may not be preserved with standard staining approaches. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.