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Paul G. McMenamin; Optimal Methods for Preparation and Immunostaining of Iris, Ciliary Body, and Choroidal Wholemounts. Invest. Ophthalmol. Vis. Sci. 2000;41(10):3043-3048.
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purpose. Investigations into the biology of resident and infiltrating immune
cells in the uveal tract of the rodent eye have been greatly aided by
the use of tissue wholemount methods. These methods offer a number of
advantages over conventional histological and frozen section
techniques. The purpose of this article is to provide a detailed step
by step guide to aid others who may wish to use this method. methods. A detailed description of whole-body perfusion fixation, dissection and
isolation of the iris-ciliary body from the anterior segment and the
choroid from the posterior segment is provided. In addition, the
techniques used to handle whole tissue pieces during single and double
immunohistochemical staining protocols, as well as the staining
protocols themselves, are described. results. In refining the techniques described, the author has catalogued a
number of frequent problems which compromise immunohistochemical
staining results. A troubleshooting guide aimed to help identify the
cause of common problems and with some suggested remedies is provided. conclusions. Although tissue wholemounts are frequently used in retinal research, a
similar approach to investigating the components of the uveal tract has
only recently been applied. The methods described in this article will
provide sufficient detail for other investigators to obtain maximum
benefit from this alternative approach and provide an additional
technique to assist in their investigations of ocular immunobiology.
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