Browsing by Author "Warden, Cassandra"

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    Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence
    (MyJove Corporation, 2021-07-05) Warden, Cassandra; Simmons, Alan J.; Pasic, Lejla; Pitzer, Ashley; Davies, Sean S.; Layer, Justin H.; Mernaugh, Raymond L.; Kirabo, Annet; Medicine, School of Medicine
    Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.