can anyone please help me how can i count activated astrocytes and microglia by using image j or any other software for statistical analysis?
thanks in advance...
Hi there, not sure if this will help or if this would work for you, but this is what I do for quantifying astrocytes and microglia.
In ImageJ, you can grayscale your images and then obtain a % readout for positive staining, on which you can then perform statistical analysis....
- Drag your image file (.jpg, .tiff etc) into ImageJ
- Image > Type > RGB Stack (the image then changes to black and white)
- Image > Adjust > Threshold (you can then adjust the threshold so that only positive staining is black)
- Analyze > Set Measurements > make sure "Area Fraction" is checked
- Analyze > Measure (a table of results will then pop up and the figure you want is the "% Area" value - this tells you what % of the image is positively stained)
You can then use this % value to quantify positive staining. Some might consider this too objective, as it is up to you to decide what is positive staining, but glial cells are so distinctive that it's pretty obvious what is positive and if you blind your images, it reduces bias as well.
I hope this helps at all, good luck!
Paul
Hi Zyed,
In addition to what Prof Peter said you will find this article educative. i use it in my case
i hope you find it beneficial
In addition to the measurement of area immunostained, which can be done for each cell type, quantitative measures that indicate progression of activation are shown in the two attached paper and include:
- proliferation indicates further activation of astrocytes
- morphological transition indicates further activation of microglia
- further definition of activation in microglial populations can be done with markers for M1 and M2 phenotypes (refer to other labs and papers)
We do stereological quantification of microglia stained with Mac-1 (or Iba-1) and while counting we classify also cell morphology. This can be applied to: rats, mice and monkeys.
In addition as suggested different proteins should be used in parallel to get a better idea of the pehotype.
See:
Article Long-term polarization of microglia upon α-synuclein overexp...
Article Microglia Acquire Distinct Activation Profiles Depending on ...
To count cells, any cell you can stain immunologically, you can use stereoinvestigator software, it is the gold standard to do this procedure. To classify astrocytes and microglia as activate or not, is a very difficult task without expression data, to know what kind of molecules it is expressing. We have some knowledge about the link between morphology and astrocytic and microglial function, but this is not accurate.
Best regards and good luck.
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