Abstract：
H vessels are an essential link in angiogenic-osteogenic coupling and orchestrate the process of bone healing. H vessels are critically deficient in the setting of radiation-induced fractures, which have been reported to occur in up to 25% of patients undergoing radiotherapy. By increasing H-vessel proliferation, Deferoxamine (DFO) revitalizes the physiologic response to skeletal injury and accelerates irradiated fracture repair. H-vessel quantification is therefore an important outcome measure in histologic analysis of bone healing. However, an optimized protocol for staining H vessels in formalin-fixed paraffin-embedded (FFPE) tissue sections has not been reported. With this protocol, we describe a method of staining FFPE bone samples with minimal background fluorescence and high signal-to-noise ratio. We examined mandibular specimens in a rat model of bone healing from a range of fracture conditions, including healthy bone (Fx), irradiated bone (XFx), and irradiated bone with DFO treatment (XFx-DFO). Quantitative analysis revealed a significant increase of H vessels in the XFxDFO group compared to both the Fx and XFx groups. By optimizing immunofluorescent staining of H vessels in FFPE samples across a range of fracture conditions, we offer investigators an efficacious means of producing reliable imaging for quantitative analysis of H vessels in an irradiated fracture callus.
摘要：
H血管是血管生成-成骨耦合的重要环节，并协调骨愈合过程。H血管在辐射诱导骨折的设置中严重不足，据报道，在接受放疗的患者中，有高达25％的患者发生这种情况。通过增加H血管增殖，去铁胺（DFO）可恢复对骨骼损伤的生理反应，并加速辐照骨折修复。因此，H血管定量是骨愈合组织学分析中的重要结果指标。然而,在福尔马林固定石蜡包埋（FFPE）组织切片中染色H血管的优化方案尚未报道。有了这个协议，我们描述了一种具有最小背景荧光和高信噪比的FFPE骨样品染色方法。我们在一系列骨折条件下的骨愈合大鼠模型中检查了下颌骨标本，包括健康骨骼（Fx），辐照骨（XFx），和用DFO处理(XFx-DFO)照射的骨。定量分析显示,与Fx和XFx组相比,XFxDFO组中的H血管显著增加。通过在一系列骨折条件下优化FFPE样品中H血管的免疫荧光染色，我们为研究人员提供了一种有效的方法，可以产生可靠的成像，以定量分析辐照的骨折骨痂中的H血管。
