Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize
IgG and Nephrin staining, and in turn, our understanding of MCD.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45552 |
| Date | 31 January 2023 |
| Creators | Ghafwari, Jamail |
| Contributors | Henderson, Joel M., Duffy, Elizabeth R. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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