An antibody panel for immunohistochemical analysis of the retina in Davidson's-fixed, paraffin-embedded eyes of rats

• Published in: Experimental and toxicologic pathology : official journal of the Gesellschaft für Toxikologische Pathologie Vol. 61; no. 2; pp. 91 - 100
• Main Authors: McKay, Jennifer S., Steele, Samantha J., Ahmed, Gulshanara, Johnson, Elizabeth, Ratcliffe, Kerry
• Format: Journal Article
• Language: English
• Published: Munich Elsevier GmbH 01-03-2009; Elsevier
• Subjects: Animals; Antibodies, Monoclonal - immunology; Apoptosis; Biological and medical sciences; Biomarkers - analysis; Caspase 3 - immunology; Cell Proliferation; Cleaved caspase-3; Davidson's fixative; Eye - chemistry; Eye - immunology; Eye - pathology; Glial fibrillary acidic protein; Glial Fibrillary Acidic Protein - immunology; Glutamate-Ammonia Ligase - immunology; Glutamine synthetase; Histones - immunology; Immunohistochemistry; Immunohistochemistry - methods; Investigative techniques, diagnostic techniques (general aspects); Male; Medical sciences; Paraffin Embedding; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Phospho-histone H3; Rat eye fixation; Rats; Rats, Long-Evans; Retina; Rhodopsin; Rhodopsin - immunology; Sensitivity and Specificity; Staining and Labeling; Synaptophysin; Synaptophysin - immunology; Immunohistochemistry; Synaptophysin; Rat eye fixation; Rhodopsin; Phospho-histone H3; Glial fibrillary acidic protein; Davidson's fixative; Cleaved caspase-3; Retina; Glutamine synthetase; Rat; Cysteine endopeptidases; Paraffin; Eye; Visual system; Ligases; Glutamate-ammonia ligase; Carbon-nitrogen ligases; Enzyme; Antibody; Rodentia; Peptidases; Vertebrata; Anatomic pathology; Mammalia; Fixation; Animal; Hydrolases; Histone H3
• ISSN: 0940-2993; 1618-1433
• DOI: 10.1016/j.etp.2008.06.005

Unlike most other tissues, the optimal fixative for preserving eye morphology is considered to be Davidson's fixative or modified Davidson's rather than formalin. However, the methodology for antibodies to be used in tissues fixed this way is not normally outlined in current antibody datasheets. Additionally, where eyes have been stored in Davidson's fixative, the efficacy of retrospective analysis of eye morphology by immunohistochemistry is largely unknown. The aim of this study was to compare a panel of six antibodies in both Davidson's-fixed and formalin-fixed pigmented and non-pigmented rat eyes, in order to provide optimal methods for future retinal immunohistochemical evaluation with image analysis. The antibodies evaluated were raised against rhodopsin, synaptophysin, glutamine synthetase, glial fibrillary acidic protein (GFAP), cleaved caspase-3 and phospho-histone H3 (PH3). Overall, the staining quality of these antibodies was found to be optimal in Davidson's compared to formalin-fixed tissues after a time period of up to 4 days in fixative. The methods outlined thus provide a platform for future detailed analysis of retinal pathology in Davidson's-fixed eyes.