![]() ![]() Studio software, Hamamatsu NZ Acquire and NDP view 2, Duolink Image Tool). Statistical significance was calculated with one-tailed, two-sample unequal variance t test. State-of-the-art microscopy is a vital tool in understanding the critical. Obtain objective quantification using Duolink. Mean values ± SEM from quantifications of at least three cells from three different experiments are shown. Single protein interactions visualized using fluorescence and brightfield respectively. Quantification of the PLA signals was done using the Duolink image tool ( g). A typical image of the PLA signals in the presence of competitor is shown without ( e) and with ( f) phalloidin stain. d Schematic of the setup for competition of the PLA signal in the presence of excessive amounts of the free γ-secretase inhibitor L-685,458. 4, and show that it abrogates GAPDH-Siah-1 PLA complex under the conditions tested. The Duolink ImageTool software can be installed and used on computers with the following 32-bit or 64-bit operating systems: Windows XP, Windows Vista, or. Next, we used R-(-)-Deprenyl (deprenyl), a known inhibitor of GAPDH. The nuclei are automatically detected and cytoplasm size estimated, enabling single. c Same image as in b but with phalloidin stain ( gray) to visualize also the cell structure. We quantify the PLA signal using Duolink Image Tool and observe a clear enhancement of GAPDH-Siah-1 PLA signal upon treating the cells with GSNO or SNAP. Use Duolink ImageTool to obtain objective quantification of PLA signals. Images of in situ PLA stained cell cultures were analyzed with the freeware software Blobfinder (Allalou and Wahlby, 2009) or with Duolink ImageTool (Olink. ![]() Images were imported in merged tiff formats containing both signal and nuclei channels. Quantification of PKA-dependent channel phosphorylation (as PLA signals per cell) was performed using the Duolink Image Tool software (Sigma-Aldrich). b Typical confocal image showing the PLA signals ( red dots) representing the association between active γ-secretase and MAO-B and DAPI staining to visualize nuclei ( blue). Images were captured and processed identically across all experimental groups. a Schematic drawing of the PLA used to study the association between active γ-secretase and MAO-B using GTB as a PLA probe. In-situ proximity ligation assay ( PLA) in cultured neurons showing association between MAO-B and active γ-secretase. ![]()
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