Can anyone recommend a good clean western blotting antibody for human P2X3? It looks like most of the widely-used antibodies for rodent P2X3 don’t work in human.
I don’t have any specific experience but this is a good resource:
https://www.citeab.com/antibodies/search?q=p2x3
Looks like the Alomone one detects human
But my personal experience with Alomone abs doing a Western-like assay (Wes, Protein Simple) has been poor. Cell Signaling antibodies are almost always a win, sometimes Millipore. Worth a try. See if you can get some trials.
Thanks! We just purchased the Alomone antibody and it didn’t work for mouse IHC or human western on the first try, but we will do some trouble-shooting to see if we can’t get it to work. I will post results if we succeed.
Honestly that’s been my experience with Alomone. Haven’t really gotten one to work yet for any assay, although I see citations showing that they can work. Do you need protein level detection or could you do an RNAscope? You should see if you can get money back for the Alomone if you did it with an approved application (mouse IHC).
Sorry I saw this post so late.
The P2X3R antibody from Santa Cruz is excellent on human DRG and spinal cord with IHC (I havent tried WB but their datasheet looks promising):
I use it with an isotype (IgG1) specific secondary antibody and its always beautiful.
Here is my paper using it on hDRG and spinal cord:
Thanks Stephanie! I’ll give it a shot.
Another question related to IHC in human tissues: given the extensive autofluorescence issues, do you think it is better to use colorimetric IHC (HRP and AP) for staining? Or are their challenges with that approach too?
@dmolliver @sshiers papers have a lot of fluoroscence imaging. The multiplex capabilities and subcellular localization are major advantages. But you’re right, AF is a problem. We have explored quenching methods. We’ve had success with this in our lab:
I agree with @achamess. True black is excellent and I’ve switched to using it with all human tissue IF experiments. Immunoperoxidase IHC is also a great idea as you suggest, but it does limit multiplexing.
(In the above paper, I didnt use True Black in the human DRG experiments since I did those experiments before I was introduced to True Black. However, in the spinal cord experiments I used it and you can see the negative control is truly negative. Bottom panel).
The stock comes at 20X. I dilute it to 1X in 70% Ethanol before use. I spin it down and try to pipette from the top of the tube since it has a precipitate that can sit on the sections and obscure the view while imaging.
I make about 200uL per experiment since a little bit goes a long way. After I finish my IHC, I tap or Kim wipe off any excess PBS from the sections and then cover them with True Black for 1 minute. I do this 1 slide at a time to be consistent with the timing. After the 1 minute, I use a squirt bottle filled with Milli-Q water and create a a constant stream of water down the side of slide to remove the excess TrueBlack and make sure any of the precipitate floaties comes off. Air dry and coverslip as normal.
It is super easy and works beautifully. The lipofuscin is still there, but it is turned black so it doesnt show up in imaging.
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