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To get the best staining pattern, it is worth conducting an up-front literature research about protein localization and cell confluency. At time of staining, the cell density should be around 50%. When cell density is too high, cell architecture is deformed and may effect higher background at low magnifications. When cell density is too low, it is more challenging to find the field with the optimal cell pattern.
Seed cells in appropriate volume of media in 12- or 24- well plates on coverslips.
Let them grow until the desired cell density is reached.
If you are performing any stimulation experiments, recalculate cell number and time to reach cell density needed after the stimulation experiment.
|Tip: Know your protein of interest. For instance, stainings for junction proteins need the appropriate cell confluency in order to represent the cell–cell contacts. Different cell densities might show different results here.|
Sterilize coverslips using an autoclave.
Wash coverslips briefly with ethanol in a laminar flow hood.
Let coverslips dry completely before use.
Add coverslips to the well plate using tweezers.
Some cell types may not attach well to glass coverslips.
Special Coating Circumstances
Some tissues (e.g., blood vessels or brain samples) may require gelatin-coated or poly-lysine-coated slides to remain attached to the slide after multiple wash steps.
Add an appropriate volume of coating solution to the coverslip, covering the whole surface. Rock the well plate smoothly in order to ensure equal coating of the surface.
Incubate the coating solution according to the manufacturer’s instructions (time, temperature).
Thoroughly wash the surface with 1x PBS or sterile cell culture grade water.
When cells have reached the desired cell confluency, aspirate the cell culture media and wash 2x PBS.
Add fixative to each well, making sure the surface is well covered. Incubation time depends on the fixative used.
Remove the fixative and wash 2x PBS.
If not immediately proceeding to antibody staining, leave some PBS in the well and store the sealed plate at 4°C. Fixated cells can be stored for a few weeks in the cold. Make sure the coverslips don’t dry out.