%0 Journal Article
%T Optimizing immunofluorescent staining of H vessels within an irradiated fracture callus in paraffin-embedded tissue samples.
%A Sheppard NT
%A Daniel MC
%A Nelson NS
%A Donneys A
%A Buchman SR
%J J Histotechnol
%V 0
%N 0
%D 2024 Jul 3
%M 38957981
%F 1.918
%R 10.1080/01478885.2024.2371060
%X 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.