Optic nerve tissue was collected and processed in accordance with previously described protocols [20 (link),33 (link),34 (link),67 (link)]. Two weeks or three months following injury or sham procedures, rats were euthanized with Euthal (Pentobarbitone sodium, 850 mg/kg; Phenytoin sodium, 125 mg/kg; i.p.) and transcardially perfused with 0.9% saline, then 2% paraformaldehyde/2.5% glutaraldehyde/2% sucrose in 0.1M phosphate buffer (pH 7.2). Dissected nerves were stored in 0.13M Sorenson’s phosphate buffer (pH 7.2). Optic nerves were further cleaned under a dissection microscope to remove excess tissue and the dura sheath, taking care to avoid distortion or stretching of the nerve.
Cleaned and trimmed optic nerves were postfixed in 1% osmium (Electron Microscopy Sciences, ProSciTech, Townsville, QLD, Australia: Cat#C011). A Lynx processor was used to dehydrate the tissue through an ethanol series to propylene oxide and tissue was then infiltrated with resin into Araldite Procure mixture (ProSciTech, Townsville, Queensland, Australia: Cat# 039). Epoxy resin-embedded tissue segments were cured for 24 h at 60 °C and serially sectioned on an ultramicrotome (LKB Nova, Bromma, Sweden). One µm transverse sections were deplasticized with saturated NaOH in 70% (v/v) ethanol and stained for 15–30 s at 95 °C in aqueous toluidine blue in 1% borax. The transverse nature of the sections was confirmed by the circular appearance of the axons. Low-power micrographs of entire sections were taken at 20× magnification to identify the injury site along the optic nerve for transmission electron microscopy (TEM) analysis. Transverse ultra-thin sections (100 nm) of optic nerve at the injury site were then cut using a diamond knife, mounted onto copper support grids (3.05 mm), and poststained with uranyl acetate and lead citrate [33 (link),67 (link)].
Cleaned and trimmed optic nerves were postfixed in 1% osmium (Electron Microscopy Sciences, ProSciTech, Townsville, QLD, Australia: Cat#C011). A Lynx processor was used to dehydrate the tissue through an ethanol series to propylene oxide and tissue was then infiltrated with resin into Araldite Procure mixture (ProSciTech, Townsville, Queensland, Australia: Cat# 039). Epoxy resin-embedded tissue segments were cured for 24 h at 60 °C and serially sectioned on an ultramicrotome (LKB Nova, Bromma, Sweden). One µm transverse sections were deplasticized with saturated NaOH in 70% (v/v) ethanol and stained for 15–30 s at 95 °C in aqueous toluidine blue in 1% borax. The transverse nature of the sections was confirmed by the circular appearance of the axons. Low-power micrographs of entire sections were taken at 20× magnification to identify the injury site along the optic nerve for transmission electron microscopy (TEM) analysis. Transverse ultra-thin sections (100 nm) of optic nerve at the injury site were then cut using a diamond knife, mounted onto copper support grids (3.05 mm), and poststained with uranyl acetate and lead citrate [33 (link),67 (link)].
Free full text: Click here
