Computational protocol: Macrophage scavenger receptor SR-AI contributes to the clearance of von Willebrand factor

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Protocol publication

[…] A detailed description of the microscopic analysis is provided in the Online Supplementary Material. Briefly, after saturation of nonspecific binding sites, cells were exposed to primary antibodies for 2 h at room temperature, followed by incubation for 1 h with secondary antibodies. Nuclei were counterstained with 4′,6′-diamidi-no-2-phenylindole. Alexa-Fluor647 or Alexa-Fluor488-labeled phalloïdin was used to determine cell boundaries.For the Duolink-Proximity Ligation Assay (Duolink-PLA) to detect close proximity between different proteins, double immunostaining was performed as described above with the second antibodies replaced by PLA probes (Sigma-Aldrich). The remainder of the protocol was conducted according to the manufacturer’s recommendations and the 550 nm wavelength detection kit was used. Hybridization between the two PLA probes leading to the fluorescent signal only occurs when the distance between the two detected antigens is less than 40 nm.Images were analyzed using ImageJ software for quantification of fluorescence by measuring the total pixel intensity per cell. Duolink-PLA experiments were analyzed using BlobFinder software (Uppsala University, Sweden) to quantify the number of fluorescent spots per cell. All images were assembled using ImageJ software. […]

Pipeline specifications

Software tools ImageJ, BlobFinder
Application Microscopic phenotype analysis
Organisms Mus musculus
Diseases von Willebrand Diseases
Chemicals Calcium