Encyclopedia of Experiments: Immunology
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Transcript
Take a multi-well plate containing mammalian cells encapsulated in a hydrogel system mimicking the natural extracellular matrix environment.
Remove the culture medium. Treat with a fixation buffer to crosslink the proteins, preserving cellular integrity.
Add a solution containing detergent to permeabilize the cellular membrane.
Introduce a blocking solution to prevent non-specific antibody binding in subsequent steps.
Add a primary antibody cocktail that enters cells and binds specifically to cytoplasmic intermediate filaments and the target transcription factor.
Remove the unbound antibodies. Introduce fluorescently labeled secondary antibodies and a nuclear stain to label the DNA fluorescently.
Remove the unbound molecules.
Mount the hydrogel culture onto a slide using a mounting medium.
Use a confocal microscope to observe the immunostained cells.
