Hi y'all,
I'm wondering if all the Iba-1+ cells with ameboid morphology in the inflamed rat brain are activated microglia, or if some are neutrophils. What I believe to be the original paper (J Clin Invest. Jun 1995; 95(6): 2954–2962.) showed expression by macrophages and neutrophils in cardiac allografts. Has anyone costained Iba-1 with a neutrophil marker in inflamed rat brain? We had a student who stained for Iba-1 thinking it was exclusive to microglia/macrophage, as it is commonly used to differentiate them from neurons and astrocytes in noninflammatory conditions. Since the student didn't costain with anything else useful, I'm wondering if some neutrophils are mixed in there.
It is my understanding that normally neutrophils would not cross the blood-brain barrier. If you suspect that some of the staining comes from neutrophils, you can do vascular stain and is if they are associated with blood vessels.
Thanks for your response Rad! In this case the BBB most certainly has been disrupted by the TBI, and neutrophils can and do infiltrate the area around the injury site. Vascular stain sounds like a great idea for future experiments. Right now I am primarily interested in interpreting immunofluorescent staining that has already been completed.
If you doubt that some of Iba1 -positive cells are neutrophils just check the presence of two or three nuclei in the soma of cells at the high magnification of images, why not?
Thanks Daniel for taking the time to post references!
Seems that the "microglial" markers CD11b, isolectin B4, and CD68 are definitely staining neutrophils in inflamed rat brain, as seen in the 2nd reference you provided. We should all take note of this.
In your 1st reference, the authors show some interesting differences between CD11b and Iba-1 staining in inflamed rat brain but do not appear to have seen the 1995 paper I mentioned in my question that showed Iba-1 (or rather AIF1 as they call it - it was only discovered later that Iba-1 and AIF1 are the same thing) expression in neutrophils in cardiac allografts. Instead, they claim that Iba-1 is not expressed in neutrophils by some "data not shown" in which all the MPO+ cells were Iba-1neg, and by citing this paper: Imai Y, Ibata I, Ito D, Ohsawa K, Kohsaka S. 1996. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun 224:855–862.
So, some believe Iba-1 is NOT expressed in neutrophils in inflamed rat brain.
Look into Holzer, Thomeer and Marani and others: after avulsion you will find bloodcells in the spinal cord. Electron microscopy will give you a clear answer in your case. Be aware that not all Ab are specific for neutrophils see Pivneva.
I agree with Tatyana, try to stain it and look at the nuclear morphology.
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