I am facing problems while counting Iba 1 positive cells in mouse brain section. I cut the brain slices in 20 micron thickness and performed immunohistochemistry. And now I have to perform the quantification of Iba 1 positive cells in the sections. A senior researcher just suggested to count all cells present in the photographs of the both cortex and striatum. But I think this is not the appropriate way of counting. For your convenience, I have attached a photograph here. Could you please let me know how many Iba1 positive cells are present there? Remember the thickness of the slice is 20 micrometer.
Hello all
I count all type of cells using stereological techniques. Based on stereology rules, it is best to start with tissues that are 40um. why? becuase tissue get dihydrated 50%, so you end up with 20um thickness of your tissue once it have been stained IHC. then, I use either optical fractionator probe in my software or the cavalieri probe. If you dont have access to a software like that, you can do count of 1 in every 6 sections, for a total of 8 to 10 section per animal. Do a total count in a 5 ROI of 50um by 50um or 100 um by 100 um depends on how big is the hole area you want to count then average the count. the bigger the area the bigger the ROI.
If interested in more details, you are welcome to look at my publications.
i hope this helps.
thanks all
Sandra
Hi Mohamed
I also want to count IBA1+ cells.
But I still did not cut the material.
Have any problem cut with 20 micrometer?
Hello all
I count all type of cells using stereological techniques. Based on stereology rules, it is best to start with tissues that are 40um. why? becuase tissue get dihydrated 50%, so you end up with 20um thickness of your tissue once it have been stained IHC. then, I use either optical fractionator probe in my software or the cavalieri probe. If you dont have access to a software like that, you can do count of 1 in every 6 sections, for a total of 8 to 10 section per animal. Do a total count in a 5 ROI of 50um by 50um or 100 um by 100 um depends on how big is the hole area you want to count then average the count. the bigger the area the bigger the ROI.
If interested in more details, you are welcome to look at my publications.
i hope this helps.
thanks all
Sandra
Hi, so are you guys characterizing ameboid (activated) microglia as big somas, and stubby processes?
Yes Partha,
The microglia that have a bit rounded shape, less processes and big soma are the activated or ameoboid one and that they have longer processes with smaller soma are ramified one.
Unbiased stereology using Optical fractiontor probe will be your best approach to get close estimate of Iba-1 positive microglia. Other, way of counting is to check microglia per section per area using serial sections but will give you gross estimate.
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