Immuno-Labeling of Yes-associated Protein in the Crystalline Lens
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The Ohio State University / OhioLINK
2016
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1460499774 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu14604997742021-08-03T06:35:32Z Immuno-Labeling of Yes-associated Protein in the Crystalline Lens Grant, Edwin Arthur Ophthalmology Medicine Immunology Biology Anatomy and Physiology Immunohistochemistry Immunofluorescence Yes-Associated Protein YAP Crystalline Lens Purpose: Yes-associated protein (YAP) is a transcriptional co-activator responsible for increasing cellular proliferation in multicellular tissues. YAP activity has been linked to many physiological changes throughout the body and is hypothesized to play a role in crystalline lens physiology and in cataractogenesis. Cataracts and secondary cataracts (posterior capsular opacification that occurs after cataract surgery) are the leading causes of blindness worldwide and pose major social and health related problems. A better understanding of cataractogenesis could lead to the development of novel strategies to prevent cataracts and the improvement of cataract surgical outcomes. This research aimed to develop a protocol for immunostaining YAP within the canine crystalline lens, with a long-term goal to better understand the role YAP plays in the lens in health and pathology. Methods: Starting from a standard immunohistochemical (IHC) protocol, the following steps were evaluated and optimized: antigen retrieval, antibody selection, antibody concentration, tissue blocking, tissue fixation, and tissue mounting. Furthermore a protocol for immunofluorescence (IF) was optimized in addition to further evaluations on tissue mounting and fixation.Results:Several IHC protocols tested were found to produce positive staining of YAP proteins in situ. Predominantly, cytoplasmic expression of YAP was noted in the equatorial cells within the transition zone for lens fiber differentiation in IHC experiments. Use of IF techniques in cultured lens epithelial cells (LEC) also produced positive staining for YAP. Immunoreactivity was both cytoplasmic and nuclear in cultured LEC in IF protocols; though the majority was cytoplasmic, some nuclear immunopositivity was apparent within cells that were less confluent. Western blots confirmed antibody specificity. Conclusions:This research demonstrates that YAP proteins can be evaluated within the canine lens via IHC and IF methods, as well as western blotting. Staining patterns observed in this study suggests YAP activity in normal lens physiology, such as fiber cell differentiation. The methods developed in this research can subsequently be used to compare YAP expression and function in health and pathophysiology, with a goal of developing methods to delay cataractogenesis. 2016-09-23 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1460499774 http://rave.ohiolink.edu/etdc/view?acc_num=osu1460499774 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Ophthalmology Medicine Immunology Biology Anatomy and Physiology Immunohistochemistry Immunofluorescence Yes-Associated Protein YAP Crystalline Lens |
| spellingShingle |
Ophthalmology Medicine Immunology Biology Anatomy and Physiology Immunohistochemistry Immunofluorescence Yes-Associated Protein YAP Crystalline Lens Grant, Edwin Arthur Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| author |
Grant, Edwin Arthur |
| author_facet |
Grant, Edwin Arthur |
| author_sort |
Grant, Edwin Arthur |
| title |
Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| title_short |
Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| title_full |
Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| title_fullStr |
Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| title_full_unstemmed |
Immuno-Labeling of Yes-associated Protein in the Crystalline Lens |
| title_sort |
immuno-labeling of yes-associated protein in the crystalline lens |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2016 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1460499774 |
| work_keys_str_mv |
AT grantedwinarthur immunolabelingofyesassociatedproteininthecrystallinelens |
| _version_ |
1719439675947483136 |