Tiam Mana Saffari

154 CHAPTER 7 to 1:2000 in Background Reducing Diluent (BRD, Dako, CA, USA) and incubated for 60 minutes. Secondary antibody used for CD34 visualization was Alexa Goat-Anti-Rabbit 488 (Fisher Scientific), diluted at 1:200 in BRD; slides were incubated for 60 minutes. This was followed by PGP9.5-Alexa 568 conjugate diluted in BRD at 1:50, incubated for 60 minutes. PGP9.5 primary antibody (rabbit polyclonal; Dako) was conjugated to Alexa 568 using the Zenon-Alexa 568-Rabbit IgG kit (Fisher Scientific) 15 minutes prior to incubation. Counterstain was performed using Hoechst 33342 (Fisher Scientific) for 10 minutes, to stain cell nuclei. Slides were coverslipped with an aqueous based ProLong Gold antifade mounting media (Fisher Scientific). Images of the stained slides were obtained using a f luorescence laser confocal microscope (Zeiss LSM 780, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The total nerve area was calculated by the total combined area of tissues positively stained for any of PGP 9.5, CD34 and Hoechst (staining cell nuclei) per nerve section. Images (N=8/group/time point) were analyzed using ImageJ software. REFERENCES 1. Hundepool CA, Nijhuis TH, Rbia N, Bulstra LF, Selles RW, Hovius SE. Noninvasive Ultrasound of the Tibial Muscle for Longitudinal Analysis of Nerve Regeneration in Rats. Plast Reconstr Surg. 2015;136(5):633e-639e. 2. Shin RH, Vathana T, Giessler GA, Friedrich PF, Bishop AT, Shin AY. Isometric tetanic force measurement method of the tibialis anterior in the rat. Microsurgery. 2008;28(6):452-457. 3. Fina L, Molgaard HV, Robertson D, et al. Expression of the CD34 gene in vascular endothelial cells. Blood. 1990;75(12):2417-2426. 4. Sun Y, Zhu L, Huang X, Zhou C, Zhang X. Immunohistochemical localization of nerve fibers in the pseudocapsule of fibroids. Eur J Histochem. 2014;58(2):2249.