Immunostaining of Cells Encapsulated in a 3D Hydrogel System

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.

First, aspirate the medium from each well. Then, wash the wells with 1 milliliter of warm DPBS. After washing is completed, add 750 microliters of fixation solution to each well. Then, incubate the 24-well plate at 37 degrees Celsius for 30 minutes. After the incubation, aspirate the fixation solution from each of the wells, and discar