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A Pajoohesh-Ganji, M Stepp; A Whole Mount Procedure to Simultaneously Identify the Label-Retaining Cells and Integrin Localization at the Mouse Ocular Surface . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3223.
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Purpose: To develop and utilize a whole mount procedure in order to visualize and correlate a relationship between (5-Bromo-2-deoxy-uridine) BrdU-label retaining cells (LRCs) and integrin expression in the limbus of mouse ocular surface. Methods: On the third day after birth, Balb/c mouse pups were injected twice per day (8 AM and 3 PM) with 25 µl of a 1 mg/ml solution of BrdU per gram body weight for 4 days. Pups were then allowed to mature for 6 or 8 weeks. For tissue sections, the eyes were frozen in OCT and 8 µm sections were stained simultaneously for BrdU and different integrins. Sections were first stained with the primary antibody for a specific integrin, followed by the appropriate secondary antibody. The antibodies were then fixed before proceeding with the BrdU visualization. For whole mounts, a suture was placed through the temporal sclera, eyes were enucleated, and fixed in a mixture of methanol and dimethyl sulfoxide (DMSO). Prior to staining, the lens and posterior tissues were discarded and the anterior segments were used to develop a double labeling method. Modifications were made to the protocol used on sections to visualize the BrdU and α9 integrin in whole mounts of anterior segments. Results: A lack of consistency in staining pattern for α9 integrin and LRCs was observed in various tissue sections. This observation was the foundation of our decision to develop the whole mount procedure. This method allowed us to determine that most of the α9 integrin positive cells reside in the nasal region, with fewer in the temporal, superior, and inferior regions. This distribution was true in both left and right eyes. In the sections, there were more LRCs present in the 6 weeks than the 8 weeks. The whole mounts showed that the LRCs were found at the limbus and were stained positively for α9 integrin. Many fewer LRCs were present compared to α9 integrin positive cells. Conclusion: A whole mount procedure was successfully developed to permit the simultaneous visualization of BrdU and integrins in the mouse ocular surface. Research supported by EY13559-01.
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