Hi there,
I've developed an algorithm in FIJI for quantifying Iba+ cells via DAB counterstain on IHC brain sections. My algorithm does a pretty good job of identifying the soma which allows for an accurate count of microglia cell bodies. However, as you can see from the attached images, I have a lot of glia where the processes are visible without cell bodies (which are probably out of focus).
For the purposes of my experiment, I'm just wanting to get cell counts of glia from Iba1 staining and I have other sections which will be stained for phenotypic markers of glia (e.g. CD11b). I've also tried using the skeletonize approach but I'm of the opinion that this is only useful for focusing on individual glia to assess morphology to infer activation state which is not the aim here since we have other markers to probe for that will give us that information.
Therefore, is simply counting the soma/cell bodies sufficient enough for quantification of microglia for purposes of publication in the future.
Thanks in advance,
David
how thick is a frozen section? if you pass a frozen DAB-stained section through xylenes and then alcohols, it shrinks (thickness is reduced from say 20 microns to 10 microns or so) and it will help you with seeing the cell bodies. Alternatively, doing thinner sections and taking few consecutive sections for counting soma only is doable.
Check out these articles:
https://www.jove.com/t/57648/quantifying-microglia-morphology-from-photomicrographs
https://www.nature.com/articles/s41598-017-13581-z
As Igor asked, what is the thickness of the section? I don't think the cell bodies are "out of focus", but are likely outside the thickness of the slice. Personally, if you are only quantifying numbers of microglia, cell bodies are what you want. For morphology (and even cell bodies), you may need thicker sections; check good quality publications to see what thickness they use.
Also... never infer "activation state" from morphology. Ever.
Hi all. Thanks for the feedback. I've managed to work up a few analyses to look at microglia coverage and quantification of cell bodies.
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