An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules
Transcrição
Take a multi-well plate containing adherent osteosarcoma cells on coverslips.
These bone cancer cells are pre-treated with a stress-inducing chemical, resulting in stress granule formation.
Stress granules are cytoplasmic membrane-less organelles comprising non-translating mRNAs, small ribosomal subunits, RNA-binding proteins, or RBPs, translation-related factors, and signaling proteins.
Discard the media and wash the cells with buffer. Fix the cells to preserve cellular morphology.
Permeabilize the cells to access intracellular targets. Add blocking proteins to prevent non-specific antibody binding.
Introduce primary antibodies that bind to specific RBPs and translation-related factors within stress granules.
Incubate with different fluorophore-labeled secondary antibodies and Hoechst dye.
Secondary antibodies bind to primary antibodies bound to the stress granule-associated proteins, while Hoechst molecules stain the DNA.
Mount the cell-containing coverslips using mounting media.
Using a fluorescence microscope, observe the cells to visualize and quantify stress granules, which appear as fluorescent foci characterized by the co-localization of associated proteins.
