Iba-1 and GFAP antibodies

Experimental Techniques Reagents and Equipment
1

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.

2

@tberta likes this Iba1 (goat) from Novus

3

If you must use two rabbit antibodies, you can also use tyramide specific amplification

Or you can consider in situ hybridization. I’m a big fan of RNAscope

4

I still likes the Iba1 made in goat from Novus (# NB100-1028) and we use the GFAP made in mouse from Millipore ( # MAB360).

5

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
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

6

@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!

7

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.

8

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

9

Hi,

We have regularly used a goat polyclonal antibody to Iba1 from 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

http://www.aveslab.com/products/glial-and-schwann-cell-markers/gfap-glial-fibrillary-acidic-protein-chicken-polyclonal-antibody-200ul/

10

I had success validating the iba-a 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.

1 Like
11

@Patrick Thanks for sharing. That’s good to hear. To clarify: You tried human neural tissue for Iba-1? IHC or WB?

12

Of course. I tested for IHC on FFPE tissue.

13

sweet! Good to know.