Dear Cinzia,

Perhaps you could upload a picture - so that we could get an idea of what your seeing.

Best,

Rohan.

Thanks Yelena for your support.

It is very important and I hope it works for my tissue and my project.

Cheers

Cinzia

Hi Cinzia, You could definitely label for differing states of activated microglia, such as M1 or M2 with IBA-1 or CD11b (only M1). You could also co-label for pro-inflammatory cytokines such as IL-1beta as Yelena stated, however, evidence is mounting that it is not just ameboid microglia that are releasing pro-inflammatory cytokines. Ramified microglia do this as well. In addition, IL-1beta is needed for memory consolidation. This would then co-label microglia and IL-1 beta, except the process memory consolidation would make this microglial cell a "good guy." Have you considered co-labelling for microglia and caspase-3? This would enable you to see which microglia are phagocytosing neurons or are contacting dying neurons. These could be classified as "bad." It would help to use IBA-1 for this, as it is known now that ramified microglia can phagocytose as well.

Hi Kimberly,

you give me a lot of interesting information!

I labelled my tissue with Iba 1 and I got all microglia types. I also tried with cd11b but never worked, could you suggest me one Ab that you know works on human CNS? and does CD11b label M1 microglia only?

By flow cytometer we looked for CD11b+/CD45low, do you think it could be consider as M1 microglia or these markers included M2 too?

Dear all,

In some articles, they claimed that in the FACS staining, CD45high cells are macrophages, while CD45low cells are microglia. Unlike macrophages, there are no M1 and M2 phenotypes existing in microglia, and only reactive (MHCII+) and resting microglia. What do you guys think? Do you have some good papers for reference? Thank you very much.

Zhilin, there are also no resting microglia. They are now said to be surveying (active) and ramified (morphological term). From what I know, CD45 is a marker for inflammation. From what I know, there are several isoforms of CD45, and you would use whichever antibody is specific to the leukocyte that you are trying to identify.

Unfortunately, I use mouse tissue and do not know if this would work in the human brain. It does work in mouse and rat cryostat, as well as mouse and zebra finch PEG. However, I use the CD11b from AbCam. I am sure that you could call TechSupport and see what tissues this antibody would cross-react with.

I had to do some troubleshooting with the CD11b. I did various antigen retrieval steps, titrated the antibody, determined concentration of Triton X, and the like... How have you fixed/how are you storing your tissue?

Kimberly, thank you very much for sharing your knowledge on microglia and macrophages with me. I am also working with mouse and I use fresh brain samples from both normal and disease mice for FACS analysis. We did see some CD45high cells and I am not sure whether they are activated microglia or macrophages. Is it possible to separate activated microglia from macrophages just by flow cytometry? CD45high cells as macrophages, and CD45low cells as microglia. In microglia, one can use some other markers such as MHCII to differentiate active and ramified microglia? Am I right?

Hi Zhilin,

Unfortunately my knowledge of flow cytometry is limited. I make my distinctions between types of microglia using double label IHC or by looking in-depth at cell morphology. I know that there are transgeneics where monocytes will express GFP. You could co-label for CD11b and then exclude those that also express GFP. MHC class II structures are only expressed by activated microglia. They are APC that present antigen to the helper-Tcells. You could look for the expression of the accessory molecules for antigen presentation (CD80/86). MHC class II labeling would only tell you the number of microglia that are presenting antigen. It would not tell you all numbers of activated microglia and it would not tell you about ramified microglia, to the best of my knowledge. It only tells you about activated microglia that are expressing an active MHC II R. Someone correct me if I am wrong.

Kimberly