Abstract: PO0309
Feature-Rich Covalent Stains for Interrogation of Kidney Tissue
Session Information
- Bioengineering
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Mao, Chenyi, University of Washington, Seattle, Washington, United States
- Lee, Min Yen, University of Washington, Seattle, Washington, United States
- Vaughan, Joshua C., University of Washington, Seattle, Washington, United States
Background
Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses significant challenge to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, they often suffer poor penetration in thick tissues, loose binding in heavily fixed or processed specimens (e.g., FFPE tissue), high cost, and inconsistent reproducibility or commercial availability. Thus, it would be highly useful to develop a simple yet robust labeling alternative that could rapidly produce even staining for thick tissues and be compatible with a wide range of sample processing or clearing methods.
Methods
We use conventional fluorescent dyes to covalently label abundant chemical functional groups on kidney tissues. These include the use of amine-reactive dyes for proteins and aldehyde-reactive dyes for carbohydrates. We term this approach Fluorescent Labeling of Abundant Reactive Entities (FLARE).
Results
We first showed FLARE’s utilities in freshly fixed mouse kidney tissues (~100 μm) using super-resolution fluorescent microscopy. Within glomeruli, the carbohydrate stain specifically labeled the basement membranes of the capillary loops and the mesangial matrix, while the amine stain outlined cell boundaries and the intricate details of interdigitated podocyte epithelial cells that are a major component of the glomerular filtration barrier (Fig. A). In proximal convoluted tubule, the basement membrane was also labeled by the carbohydrate stain, and the amine stain revealed mitochondria and brush border microvilli (Fig. B). Then we stained optically cleared FFPE human kidney tissues (~50 μm) without performing antigen retrieval (Fig C), revealing more general features. Furthermore, FLARE does not perturb antigenicity, where immunolabeling of proteins can be easily integrated afterwards (data not shown).
Conclusion
We have shown that FLARE reveals abundant details in a wide range of kidney tissue processing methods using super-resolution and cleared-tissue microscopy, and is compatible with other staining modalities.
Funding
- NIDDK Support