This article describes a protocol for immunostaining mammalian cells encapsulated in a hydrogel system. The method preserves cellular integrity while allowing for the visualization of specific proteins using fluorescent antibodies.
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 discard the fixative in a hazardous waste container. Next, add 1 milliliter of DPBS to the sample and immediately discard the DPBS into a hazardous waste container.
Next, add 750 microliters of permeabilization solution to permeabilize the sample for one hour at room temperature on the rocker, at 15 revolutions per minute. After one hour, aspirate the permeabilization solution, and add 750 microliters of blocking solution to the sample. Leave the sample on the rocker at room temperature for three hours. Then, dilute the desired primary antibodies with the antibody dilution solution.
Next, add 500 microliters of the antibody solution to each sample. Incubate the samples overnight at 4 degrees Celsius. The following day, aspirate the primary antibody solution. Then, wash the samples with PBST for one hour thrice, at room temperature, at 15 revolutions per minute.
Next, dilute the appropriate secondary antibodies and 5 mg/ml of DAPI stock solution to achieve a final concentration of 2.5 micrograms per milliliter. Then, add 500 microliters of the solution to each sample. Next, use aluminum foil to cover the 24-well plate and incubate the plate at 4 degrees Celsius overnight.
The next day, aspirate the secondary antibody solution and wash the samples with PBST thrice, each for 30 minutes at room temperature on a rocker. Next, position a drop of hard set mounting medium on the surface of a glass slide and place the sample on the mounting medium. Finally, use nail polish to seal the samples to the glass cover slide to prevent contamination or sample movement.
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