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B.A. Pfeffer, S.A. Bernstein, S.P. Bartels; Preservation of Structure and Immunoreactivity at the Vitreoretinal Interface of the Rabbit Eye . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5625.
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© ARVO (1962-2015); The Authors (2016-present)
To enhance preservation of vitreus components, compared to conventional fixation, and to improve analysis of the structure of cortical vitreus by means of histochemical (HC) and immunocytochemical (ICC) detection; to establish a baseline morphology for the vitreoretinal interface, preliminary to studies of induced posterior vitreus detachment (PVD).
6 pigmented rabbits underwent carotid perfusion with: 10% neutral buffered formalin + cetyl pyridinium chloride (CPC; Fixative 1); ethanol–acetic formalin + CPC (F2); or Karnofsky's + CPC (F3). Fixation of whole globes continued in each of these fixatives following enucleation. After paraffin processing and H&E staining, the degree of tissue preservation at the vitreoretinal interface was evaluated for each fixative. HC and ICC were performed on sections using antibodies for collagen II or XVIII, ABA lectin, s–100, and vimentin.
Without supplementation, standard formalin immersion fixation has historically given inconsistent preservation of vitreoretinal structures. In contrast, perfusion with all three CPC–containing fixatives yielded a well–defined cortical vitreus layer exhibiting marked antigenicity and affinity for stains, in part due to increased vitreal adherence to the inner limiting lamina (ILL) of the neural retina, from which the vitreal cortex could be differentiated by H&E staining. While F1 gave rise to better intralaminar structure, F2 caused condensation of fibrillar elements in the cortical vitreus that colocalized with profound immunoreactivity for collagen II, continuously observable from the medullary rays to the ora serrata. A prominent whorl of fibrous material with type II collagen immunoreactivity was noticeable at the anterior vitreus base. F3 appeared to produce the best differential ABA reactivity in the vitreus cortex, but also hindered detection of collagen II. Collagen XVIII immunoreactivity in the ILL was maintained with F2 only. Antigen retrieval and unmasking by means of heat and protease treatment of sectioned material amplified detection of cortical collagen II, but also compromised overall vitreoretinal structural integrity in tissue sections. The labeling of glia in the retinas from all 3 fixations was useful for distinguishing between cellular and extracellular (i.e., ILL) elements.
Histochemical techniques designed to minimize artifacts and optimize immunoreactivity provide an indispensable baseline assessment of the vitreoretinal interface.
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