Given that astrocytes have a particular morphology, sometimes in some brain regions (and particularly in human brain) cellular somata are hard to identify. My concern is to under or overestimate the number of astrocytes in human brain. On the other hand, fibers are easier to distinguish as compare with cell bodies. Then, perhaps is worthly to focus on counting fibers...
Dear Daniel. I understand your concerns.
First, I would recommend to use the bright-field microscopy and work with paraffin-embedded tissue blocks (II use anti-GFAP antibody from Biolegend, #835301 with dilution 1:1000. It works perfectly). Second, imaging should be done on a good equipment. I imaged coronal brain sections using slide scanner Axio Scan.Z1 (Zeiss, Oberkochen, Germany) with a 40×/0.95 objective recording focal distances (Z-planes) every 0.35 μm. ZEISS Apotome.2 microscope was used to capture images with a × 100/1.4 oil immersion objective.
For morphometric cell analysis you can use a free software (ImageJ for instance). I used original ZEN Blue 2.5 software (Zeiss, Oberkochen, Germany).
Anyway, a fixed background intensity threshold should be set for all sections representing a single type of staining.
Additionally, I suggest the Image-Pro® from Media Cybernetics. It is not free, however, you can ask for a demo version (for two weeks I believe). This piece of software can perform a very advanced image densitometry analysis with significantly more functions compared to ImageJ
Check, if you have Imaris from Oxford Instruments, which can be used to create a 3D image reconstruction and quantify the cell body volume, stain intensity, and the number of astroglia processes.
In my opinion, this is the best program to analyze the astrocytic cytoskeletal features. May be you can ask for a demo as well.
Good luck.
PS Take a look:
https://www.ncbi.nlm.nih.gov/pubmed/30288668
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770953/
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