To me it just looks like you are staining the cytoplasm of neurons. The dark spot in the center is the nucleus, which you mention is being stained by DAPI. Either these antibodies are non-specifically binding something in the cytoplasm of neurons, or your target proteins are being expressed in these cells. It does not look like any type of auto-fluorescence in my opinion.

Seems like non specific binding. I had it with my 3-nitrotyrosine Ab, and I could get rid of that by switching to frozen sections. I suggest that you first check whether your primaries are suitable for FFPE slices. Otherwise, you may try with frozen sections.

Alternatively, you may consider optimizing your antigen retrieval method (e.g. longer time in Citrate?-but 25min actually should be ok). You can also try another method like 2N HCl for 30 min (in that case you would need a short incubation in borat buffer to neutralize pH afterwards). Usually you would need HCl method for nucleus targeted antibodies to disrupt nuclear membrane, but sometimes it is also useful for other Abs as well. My Casp3 primary was staining every second nuclei in brain (frozen sections)- probably just because it was sticky to some non-specific target that I don't know of; that I could get rid of with HCl method. Surprising enough, it also improved my 3-nitrotyrosine staining as I got to know with a co-immunostaining.

Good luck!

Muammer

Dear Alyssa,

It is not uncommon for labeling of cytoplasmic proteins to appear as "ring-like", since those antibodies should not recognize nuclear proteins.  DAPI, of course, should be in the nucleus.

The figure with the GFAP/DAPI staining has too little contrast on my monitor for me to be sure of what I see.  GFAP is only found in the cytoplasm of astrocytes, so it is not uncommon for certain cells in a section to lack staining in the nucleus.  This would be similar to looking at an anti-neurofilament or a Nissl stain for neurons--depending on the plane of section, there is no cytoplasm above the nucleus.  Therefore, there is no staining in that area, and there appears to be a hole that is DAPI positive.  This pattern is also seen if you examine your staining with a confocal microscope, since you are looking at a very thin plane of the section.

However, I do not see any of the normal stellate (sunburst-like) staining pattern for GFAP-positive astrocytes that I normally see in brain.  I agree with Leslie that it looks more like neurons, which would not be correct.  For an example of GFAP staining, there is a confocal fluorescent image here:

https://www.mybiosource.com/prods/Antibody/Monoclonal/GFAP/datasheet.php?products_id=9200429

I also believe the staining pattern is incorrect for the von Willibrand Factor.  While this is also a cytoplasmic protein, it is found in endothelial cells, unless something causes it to leak out into the parenchyma (like a hemorrhage; example here: https://www.nature.com/articles/srep35901).  I do not see any strong staining in blood vessels in your figure, and there should be at least some near the pial surface of your section.  For an example, see Fig. 6 in this paper:

http://www.bloodjournal.org/content/bloodjournal/92/8/2791.full.pdf?sso-checked=true

As for the Iba1, I do see some ramified microglia that are labeled (they look like spiders).  Again, because this protein is cytoplasmic, the nucleus will not be stained, but the cells are so small that often the nucleus is difficult to see unless you use a nuclear marker.  I also see a few stained cells that I would interpret as activated microglia, which have much more cytoplasm.  However, I also see a lot of non-specific staining in neurons, so it is difficult to be sure--it might just be a ramified microglia located adjacent to a neuron.  You may need to play more with your protocol, either decreasing the concentration of your primary antibody, increasing the amount or time of the washes, or trying other blocking proteins.      

Are these normal brains, or animals that have had some sort of treatment?  Just curious, since there might be activated cells present.  Usually you see resting, ramified microglia in normal brain.

For an example of ramified and activated microglial staining:

http://www.sciencedirect.com/science/article/pii/S0169328X98000400

As Muammer suggested, you should check the specifications for your primary antibodies.  If you need suggestions for validated staining patterns in brains of many different animals, you can check the antibody database at the Journal of Comparative Neurology.  The link to the Excel file is here:

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1096-9861/homepage/other_resources.htm

It is always a challenge to achieve good immunolocalization of a protein.  You need really good, validated antibodies to get believable results.

Good Luck!

Jill