# Iba-1 and GFAP antibodies **URL:** \ **Category:** Reagents and Equipment\ **Created:** [October 24, 2017, 3:32pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308 "2017-10-24T15:32:19Z")\ **Posts on this page:** 13\ **Page:** 1
**Author:** ![ShanTan](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/shantan/32/242_2.png) [@ShanTan](https://forum.painresearcher.net/u/ShanTan)\ **Post date:** [October 24, 2017, 3:32pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/1 "2017-10-24T15:32:19Z")
Looking to see if anyone has had success with Iba-1 and GFAP antibodies that are NOT raised in Rabbit. Chicken? Goat? Looking to co-stain with an antibody that is rabbit polyclonal, and am having a hard time seeing as the GFAP and Iba-1 I’m used to using are rabbit as well. ---
**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:** [October 24, 2017, 7:12pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/2 "2017-10-24T19:12:45Z")
@tberta likes this Iba1 (goat) from Novus > [@Iba1 vs CD11b for microglia staining?](http://forum.painresearcher.net/t/iba1-vs-cd11b-for-microglia-staining/212/3): > > I agree with Alex - the Iba1 staining is superior and it may due to the quality of the antibodies. However, it is worthy to note that Cd11b/Ox42 is a membrane protein, whereas Iba1 is expressed in cytoplasm. Therefore, Iba1 produce a more plain and robust signal in section with small thickness. A general complain of using Iba1 is that the most popular antibody from Wako (#019-19741) is only made in rabbit, so many researchers have used Cd11b made mouse as an alternative in double staining. Howev… ---
**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:** [October 24, 2017, 7:34pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/3 "2017-10-24T19:34:40Z")
If you must use two rabbit antibodies, you can also use tyramide specific amplification > **[mp20911.pdf](https://assets.thermofisher.com/TFS-Assets/LSG/manuals/mp20911.pdf)** > > 635.14 KB Or you can consider _in situ_ hybridization. I’m a big fan of RNAscope > [@RNAscope (In situ hybridization) to study gene expression in pain-related tissues](http://forum.painresearcher.net/t/rnascope-in-situ-hybridization-to-study-gene-expression-in-pain-related-tissues/102): > > In situ hybridization is a powerful method to interrogate gene expression in tissues. It is known as being a challenging procedure due to the many steps where errors and obstacles arise. We’ve recently begun to use a commercial system called [RNAscope](https://acdbio.com/science/technology-overview) from ACD Bio that gets around many of the pitfalls of traditional ISH. I’ve used it a lot now in the spinal cord and DRG. Below is an image of the gene VGAT (Slc32a1) in the dorsal spinal cord. There is nice expression, and most importantly, it was… ---
**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:** [October 24, 2017, 8:33pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/4 "2017-10-24T20:33:37Z")
I still likes the Iba1 made in goat from Novus (# NB100-1028) and we use the GFAP made in mouse from Millipore ( # MAB360). ---
**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:** [October 24, 2017, 9:57pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/5 "2017-10-24T21:57:34Z")
Agree with Temo about GFAP. That antibody from Millipore is the standard. And if you want to also see neurons, use the fluorescent Nissl stain in blue: [https://www.thermofisher.com/order/catalog/product/N21479](https://www.thermofisher.com/order/catalog/product/N21479) 1:100 for 10 minutes is usually good You can get pretty images with multiple colors: 1. GOI (red) 2. GFAP (green) 3. Iba1 (far red) 4. Nissl (blue) More on Nissl in [brain slice](https://www.researchgate.net/post/NeuroTrace_Fluorescent_Nissl_Stain) ---
**Author:** ![ShanTan](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/shantan/32/242_2.png) [@ShanTan](https://forum.painresearcher.net/u/ShanTan)\ **Post date:** [November 23, 2017, 2:04am UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/6 "2017-11-23T02:04:43Z")
@tberta Do you have a protocol for your Iba1 goat staining? What secondary do you use? Most of my secondaries are goat anti-x AlexaFlour… What do you block with if not NGS? Thanks! ---
**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:** [November 23, 2017, 2:35am UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/7 "2017-11-23T02:35:01Z")
You’ll need anti-goat secondaries, which you can get from Life Tech. In general, I like secondaries raised in Donkey, since I’ve yet to use a Donkey primary antibody, so low probability of species-mixing problems. Temo uses a BSA buffer, as do I (he taught me). 1% BSA, 0.4% triton-x, in PBS w/ azide. That’s my all around IHC buffer now and it works for the majority of applications. ---
**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:** [November 23, 2017, 1:35pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/8 "2017-11-23T13:35:17Z")
Alex is right. BSA 1% is our default and it works pretty much for any antibody, including for the IBA-1. Briefly: Wash the slide with PBS 1x Block the slide with blocking solution (BSA 1% and Triton 0.4% in PBS 1x) for at least 30 min in RT Remove the blocking solution Apply the primary antibody diluted in BSA 1% and Triton 0.2% in PBS 1x and incubate overnight at 4°C Rinse 3x with PBS 1x on shaker (5 min each) Apply fluorophore-conjugated secondary antibody diluted in BSA 1% and Triton 0.2% in PBS 1x and incubate 1h at RT Wash 3x with PBS 1x on shaker (5 min each) Add DAPI 1:1000 for 5 min Wash with PBS 1x Mount slide with mounting media and put on slip over plate We use this secondary anti-goat antibody: [https://www.thermofisher.com/antibody/product/Donkey-anti-Goat-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-21432](https://www.thermofisher.com/antibody/product/Donkey-anti-Goat-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-21432) ---
**Author:** ![LShen](https://avatars.discourse-cdn.com/v4/letter/l/dfb087/32.png) [@LShen](https://forum.painresearcher.net/u/LShen)\ **Post date:** [January 16, 2018, 5:01pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/9 "2018-01-16T17:01:29Z")
Hi, We have regularly used a goat polyclonal antibody to Iba1 from [Antibodies.com](http://Antibodies.com) (#A82670) which stains really well. I think it is identical to the one from Novus just a lot cheaper. We also use a chicken polyclonal antibody to GFAP from Aves Labs (#GFAP). Has anyone tried a goat polyclonal antibody to GFAP? [https://www.antibodies.com/aif1-antibody-a82670](https://www.antibodies.com/aif1-antibody-a82670) [http://www.aveslab.com/products/glial-and-schwann-cell-markers/gfap-glial-fibrillary-acidic-protein-chicken-polyclonal-antibody-200ul/](http://www.aveslab.com/products/glial-and-schwann-cell-markers/gfap-glial-fibrillary-acidic-protein-chicken-polyclonal-antibody-200ul/) ---
**Author:** ![Patrick](https://avatars.discourse-cdn.com/v4/letter/p/b2d939/32.png) [@Patrick](https://forum.painresearcher.net/u/Patrick)\ **Post date:** [July 31, 2018, 5:11pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/10 "2018-07-31T17:11:40Z")
I had success validating the [iba-a](http://www.genetex.com/Iba1-antibody-GTX100042.html) antibody on a human target with GeneTex’s iba-a. I know a lot of the companies OEM a lot but I think some larger providers specifically OEM Genetex’s iba-1 and hike the price up. I may be wrong but I’m trying to account for the large price difference since Genetex is a lot cheaper and has similar/identical quality. ---
**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 31, 2018, 11:53pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/11 "2018-07-31T23:53:46Z")
@Patrick Thanks for sharing. That’s good to hear. To clarify: You tried human neural tissue for Iba-1? IHC or WB? ---
**Author:** ![Patrick](https://avatars.discourse-cdn.com/v4/letter/p/b2d939/32.png) [@Patrick](https://forum.painresearcher.net/u/Patrick)\ **Post date:** [August 1, 2018, 6:22pm UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/12 "2018-08-01T18:22:09Z")
Of course. I tested for IHC on FFPE tissue. ---
**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:** [August 2, 2018, 12:43am UTC](https://forum.painresearcher.net/t/iba-1-and-gfap-antibodies/308/13 "2018-08-02T00:43:57Z")
sweet! Good to know.