Hello all,
I stained the cells (LAD2) for immunofluorescent microscopy and mounted the slides by mounting medium with DAPI (Vectorshield). When mounting, I pressed the cover slips against the slide a little bit hard to remove the excessive mounting medium. I observed that in some slides the morphology of the cells were changes. I do not know whether the morphology changes were due to the treated drug I used (no previous report about the effect of the drug) or due to the mounting step.
Does pressing the cover slips hard cause the changes of cell morphology?
Thank you very much for helping me.
Hello, I doubt morphology alteration can occur mounting monolayer cells growing on a coverslip (or slide chamber). Often morphology changes occur during the washing and fixation steps: if you carry them on at rt or 4 °C, for example, cells often shrink and round up. Use instead 37 °C. Also a medium with wrong osmolar concentration can affect cell morphology (often having 2-4% PFA in phosphate buffer is not sufficient, so you can add 0.32M sucrose or be sure you are in PBS, i.e. with 137 mM NaCl and 2.5 mM KCl). You can also use 4% PFA and 0.25% glutaraldehyde in PBS to better preserve morphology.
Good luck
Hello
I agree, pressing hard on the coverslip will not cause this distortion. However, if the cells were not adhering very well or the cells were damaged during the staining steps, pressing hard on the coverslip will damage the cells even further.
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