I am working on inflammation in brain endothelial cells. I use LPS as inflammatory agent, then I treat my cells with a drug in order to reverse the LPS-related damage. I have three experimental groups: control, LPS-treated, LPS+drug -treated. I have recently started to perform IF stainings, how should I do the imaging? By capturing images with 10 μm z-stacks and z-projection of all images, then cell counting? Or else, should I capture the whole area with a single z, then perform analysis afterwards? Since I am new in imaging and image analysis, recommendations would be highly appreciated. Many thanks!
If you are working with cultured cells a z-stack is probably not necessary, unless you are looking for differences in subcellular organization.
Hi Irem,
The answer to your question may depend on which protein(s) you are targeting by IF staining. If you are looking at the global expression (e.g. presence/absence) or change (increase/decrease) in protein levels in your cells, then you don't need z-stacking. For this approach lower magnification power (e.g. 10-20X objectives) is preferred one (you should try to visualize as many cells as possible for quantitative analysis using IF analysis tools such as ImageJ or whichever software that comes with your confocal microscope). However, if you are also interested in detecting specific localization/relocalization of your protein(s) of interest, you could use z-stacking and higher magnification for this analysis. 10 μm z-stacks as you indicated in your message may be too high for the work in cell culture. Cells (particularly endothelial cells) in culture are very thin (0.1-5μm; with nuclei being the "thickest" part of the cell), so the z-stacks should be in the range of 0.1-0.2 um.
Hope that will help to strategize your experiments/analyses.
Best of luck!
P.S. below are the links to a couple of papers that may be of help in your experiments.
doi: 10.1038/sj.mn.7800185.
DOI: 10.1016/j.ajpath.2016.12.025
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