Dear All,
I performed an immunohistochemistry in mouse brain tissue looking for astrocytes (stained for GFAP). Now, I would like to evaluate any morphologic change between the different groups I am comparing.
I do know it would be possible with Fiji/ImageJ through Sholl analysis and I did read several papers suggesting so, but I do not understand what I am actually measuring.
Do you have any idea, protocol, suggestion on how to do the image analysis using either ImageJ or another software (I'd prefer an open-source software).
Thank you,
Salvo.
Hi Salvatore,
1. Firstly, you may need to be sure of the astrocytes in the slides. Then, capture the image using your desired microscope.
2. You will need to calibrate (scale) the captured image before analyzing your images.
3. Once calibrated, you will need to so the main analysis using the image J which involves measurement of length, cross sectional area, cell width etc. which you will further need to compare in all the groups that you have using the appropriate method of statistical analysis.
I hope this can go a way to answer your question.
You could do Sholl analysis... There are lots of papers describing Sholl analysis on neurons... I imagine you could swap neuron for astrocyte and use a similar protocol. Our lab frequently used skeleton analysis for microglia (free Image J plugging), and I imagine you could also use it to analyze astrocytes. I can't speak to the completeness of GFAP labeling of astrocyte processes, but if you already have the tissue, it could be worth trying. Good luck!
I used ImageJ, ZEN 2.5, and Imaris® for morphometric analysis of GFAP positive astrocytes.
Take a look:
https://www.ncbi.nlm.nih.gov/pubmed/31540372
https://www.ncbi.nlm.nih.gov/pubmed/30288668
If you are going to perform a large-scale analysis of 3D astrocyte morphology, I would strongly recommend Imaris. With this piece of software, you can reconstruct individual cells and analyze the number of astrocytic protoplasmic processes. The analysis of the stain intensity and cell volume can be done very easily in ZEN 2.5, which is much more powerful compared to ImageJ.
Good luck!
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