| Institution: | 1. Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA;2. Department of Ophthalmology and Vision Science, University of California, Sacramento, CA 95817, USA;1. Ophthalmology, Clinical Neurosciences Research Group, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom;2. Fight for Sight, Clinical Neurosciences Research Group, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, United Kingdom;1. Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan;2. Discovery Research Laboratories III, Ono Pharmaceutical Co., Ltd., Osaka, Japan;1. The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA;2. Center for Nanomedicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA;3. Department of Chemical and Biomolecular Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA;4. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA;5. The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA;6. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA;7. Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA;8. Department of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA;9. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA |
| Abstract: | Immunohistochemical and ex vivo anatomical studies have provided many glimpses of the variety, distribution, and signaling components of vertebrate retinal neurons. The beauty of numerous images published to date, and the qualitative and quantitative information they provide, indicate that these approaches are fundamentally useful. However, obtaining these images entailed tissue handling and exposure to chemical solutions that differ from normal extracellular fluid in composition, temperature, and osmolarity. Because the differences are large enough to alter intercellular and intracellular signaling in neurons, and because retinae are susceptible to crush, shear, and fray, it is natural to wonder if immunohistochemical and anatomical methods disturb or damage the cells they are designed to examine. Tissue fixation is typically incorporated to guard against this damage and is therefore critically important to the quality and significance of the harvested data. Here, we describe mechanisms of fixation; advantages and disadvantages of using formaldehyde and glutaraldehyde as fixatives during immunohistochemistry; and modifications of widely used protocols that have recently been found to improve cell shape preservation and immunostaining patterns, especially in proximal retinal neurons. |
