P G McMenamin1. 1. Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth. mcmenamin@anhb.uwa.edu.au
Abstract
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.
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.
Authors: Carolyn K Pan; Chandrasekar Durairaj; Uday B Kompella; Ogechi Agwu; Scott C N Oliver; Hugo Quiroz-Mercado; Naresh Mandava; Jeffrey L Olson Journal: J Ocul Pharmacol Ther Date: 2011-05-16 Impact factor: 2.671
Authors: Holly R Chinnery; Samuel McLenachan; Nicolette Binz; Yan Sun; John V Forrester; Mariapia A Degli-Esposti; Eric Pearlman; Paul G McMenamin Journal: Am J Pathol Date: 2011-11-12 Impact factor: 4.307
Authors: Irina Semkova; Florian Kreppel; Gerhard Welsandt; Thomas Luther; Jolanta Kozlowski; Hanna Janicki; Stefan Kochanek; Ulrich Schraermeyer Journal: Proc Natl Acad Sci U S A Date: 2002-09-18 Impact factor: 11.205