Proteostat aggresome detection dye

Upon ER stress, aggresomes commonly increase. Aggresomes were stained using Proteostat^® Aggresome Detection dye (Enzo Life Sciences) [61 (link)], and the degree of aggresome accumulation was determined by flow cytometry. Briefly, cells were prefixed with 4% paraformaldehyde for 30 min and permeabilized by 0.5% Triton X-100 for 30 min at 4 °C. Finally, cells were stained with aggresome staining dye (1:10,000) for 30 min, analyzed using a Guava easyCyte flow cytometer and plotted with FlowJo.

Protein aggregate levels were measured using the Proteostat Aggresome detection dye (Enzo Life Sciences ENZ-51035-K100) following the manufacturer’s instructions. The 488 nm excitable red fluorescent molecular rotor dye becomes brightly fluorescent upon binding to aggregated proteins within vesicles produced during aggresome formation. Briefly, prior to the FACS experiment, cells were washed with PBS, harvested via trypsin dissociation and fixed with 4% PFA. Next, cells were permeabilized and stained with 1:10,000 dilution of the Proteostat Aggresome detection dye in PBS + 0.1% FBS for 30 min.

Brain samples were fixed in formalin and paraffin embedded. 10-μm thick sections were obtained. After deparaffinization, we stained with thioflavin S, aggresome dye, and BTA-1. Briefly, thioflavin S (Sigma) staining was performed by incubation with a 0.1% thioflavin S solution for 10 min, followed by two washes in 80% ethanol. For BTA-1 staining, we incubated the samples with a 100 nm BTA-1 solution for 45 min, followed by a wash in distilled water. For aggresome staining, we used Proteostat Aggresome detection dye (Enzo Life Sciences) for 10 min, followed by a 20 min wash with 1% acetic acid. Samples were cover-slipped with Vectashield (Vector Labs) or Fluoro-Gel (EMS) mounting medium. Each group of samples was analyzed by immunofluorescence. Images were acquired with a Leica DMI 6000B microscope.
Confocal microscopy was performed at the Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology University of Texas, Health Science Center. Brain histological sections were visualized using a Nikon A1R Confocal Laser Microscope System. The acquisition was made at standard high-resolution confocal mode. Image analysis and quantification was performed using Image J (RSB, Reserve Services Branch, NIH).