Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts

PURPOSE. Investigations into the biology of resident and infiltrating immun e 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 advantag es over conventional histological and frozen section techniques. The purpos e 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, dissectio n 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 immunohistoche mical 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 r esults. 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 res earch, a similar approach to investigating the components of the uveal trac t has only recently been applied. The methods described in this article wil l provide sufficient detail for other investigators to obtain maximum benef it from this alternative approach and provide an additional technique to as sist in their investigations of ocular immunobiology.