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.
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
I still likes the Iba1 made in goat from Novus (# NB100-1028) and we use the GFAP made in mouse from Millipore ( # MAB360).
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:
1:100 for 10 minutes is usually good
You can get pretty images with multiple colors:
- GOI (red)
- GFAP (green)
- Iba1 (far red)
- Nissl (blue)
More on Nissl in brain slice
@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?
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.
Alex is right. BSA 1% is our default and it works pretty much for any antibody, including for the IBA-1.
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:
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?
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.
@Patrick Thanks for sharing. That’s good to hear. To clarify: You tried human neural tissue for Iba-1? IHC or WB?
Of course. I tested for IHC on FFPE tissue.
sweet! Good to know.