# IHC for glial activation **URL:** \ **Category:** Experimental Techniques\ **Created:** [July 6, 2016, 8:24pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49 "2016-07-06T20:24:01Z")\ **Posts on this page:** 7\ **Page:** 1
**Author:** ![LegakisL](https://avatars.discourse-cdn.com/v4/letter/l/8baadc/32.png) [@LegakisL](https://forum.painresearcher.net/u/LegakisL)\ **Post date:** [July 6, 2016, 8:24pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/1 "2016-07-06T20:24:01Z")
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. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [July 6, 2016, 8:30pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/2 "2016-07-06T20:30:03Z")
@LegakisL Good question. For astrocytes, you can use GFAP. See the [Antibody Database](http://forum.painresearcher.net/t/antibodies-for-pain-research-database/30) 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](https://www.bio-rad-antibodies.com/mouse-cd68-antibody-fa-11-mca1957.html) 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! ---
**Author:** ![lfqueme](https://avatars.discourse-cdn.com/v4/letter/l/f17d59/32.png) [@lfqueme](https://forum.painresearcher.net/u/lfqueme)\ **Post date:** [July 6, 2016, 8:47pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/3 "2016-07-06T20:47:44Z")
I have seen the results from the one from the iba1 from Abcam, it seems good. ---
**Author:** ![tberta](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/tberta/32/145_2.png) [@tberta](https://forum.painresearcher.net/u/tberta)\ **Post date:** [July 6, 2016, 9:39pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/4 "2016-07-06T21:39:39Z")
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](https://www.novusbio.com/AIF-1-Iba1-Antibody_NB100-1028-002mg.html) This article can be useful for your M1/M2 transition: > **[The Prevalence and Phenotype of Activated Microglia/Macrophages within the...](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663759/)** > > Cervical compressive myelopathy, e.g. due to spondylosis or ossification of the posterior longitudinal ligament is a common cause of spinal cord dysfunction. Although human pathological studies have reported neuronal loss and demyelination in the... However, I don’t have experience with and guarantee about the antibodies used (including the iNOS one) for this study. ---
**Author:** ![LegakisL](https://avatars.discourse-cdn.com/v4/letter/l/8baadc/32.png) [@LegakisL](https://forum.painresearcher.net/u/LegakisL)\ **Post date:** [July 6, 2016, 10:00pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/5 "2016-07-06T22:00:22Z")
Thank you! This is very helpful. Will probably try the sample. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [July 7, 2016, 3:17am UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/6 "2016-07-07T03:17:36Z")
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. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [July 8, 2016, 5:46pm UTC](https://forum.painresearcher.net/t/ihc-for-glial-activation/49/7 "2016-07-08T17:46:19Z")
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.](http://www.sciencedirect.com/science/article/pii/S2211124716307586)