ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

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