IHC for glial activation

I’m going to be performing some IHC of spinal cords following paclitaxel treatment. Does anyone have experience with markers of microglial activation? I will be using Iba1 as a microglial marker but does anyone have experience with iNOS or any other activation marker to compare a M1/M2 ration? What seems to work best? Appreciate the help.

@LegakisL Good question. For astrocytes, you can use GFAP. See the Antibody Database for good GFAP antibodies. For microglia, Iba1 is typically used, as you say, and in addition to the most popular rabbit Iba1 ab from Wako, @tberta also has found a good one from Abcam made in goat (see Database). Ox42 is also used for microglia, but I haven’t used it myself. Anti-CD68 from Ab Serotec is quite good. I use at a concentration of 1:1000 for macrophages in the DRG and nerve tissue. You could try in the spinal cord too, since CD68 is thought of as a marker of activated macrophages/microglia, even though resting state cells also have. @tberta knows a lot about glial activation, so hopefully he can chime in.

I haven’t used an iNOS antibody for M1/M2 transition, but hopefully someone here can suggest a good marker.

Good luck!

I have seen the results from the one from the iba1 from Abcam, it seems good.

Actually, the Iba1 antibody made in goat is from Novus Biologicals and you can have a sample size to try. https://www.novusbio.com/AIF-1-Iba1-Antibody_NB100-1028-002mg.html

This article can be useful for your M1/M2 transition:

However, I don’t have experience with and guarantee about the antibodies used (including the iNOS one) for this study.

Thank you! This is very helpful. Will probably try the sample.

Thanks for the correction @tberta

I would also add that for Iba1 and GFAP, the staining is better if you do free-floating slices (40 um) vs. on slides. Some antibodies look great no matter what, like NeuN, but for microglia and astrocytes, I’ve gotten superior staining with free-floating sections.

This very recent paper looked at a number of microglia markers with IHC, including CD169, Iba1, P2Y12. Lots of useful info here, and an important paper regarding the role of microglia.

Gu, N., Peng, J., Murugan, M., Wang, X., Eyo, U.B., Sun, D., Ren, Y., DiCicco-Bloom, E., Young, W., Dong, H., et al. (2016). Spinal Microgliosis Due to Resident Microglial Proliferation Is Required for Pain Hypersensitivity after Peripheral Nerve Injury. Cell Reports 0.

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