FoxP3 stain does not work

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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kuehla
Posts: 2
Joined: Thu Feb 28, 2019 12:37 pm

FoxP3 stain does not work

Post by kuehla » Mon Aug 19, 2019 6:56 am

The staining of FoxP3 for IMC with the clone 236A/E7 (# 3155016D) does not work in our hands. We are down to a dilution of 1:10, but still do not get a signal.
We´ve now ordered the same clone from a different company (unlabelled) and tried in IHC, where it worked fine with retrieval conditions at pH6, but not at pH9. The Fluidigm data sheet and publications though state a HIER at pH9. Is there another step needed to get a positive FoxP3 signal? We follow the Fluidigm protocol for IMC and other antibodies within the same cocktail work fine.

MariekeIJsselsteijn
Posts: 8
Joined: Mon Jan 15, 2018 10:52 am

Re: FoxP3 stain does not work

Post by MariekeIJsselsteijn » Mon Aug 19, 2019 7:09 am

We have experience with the 236A/E7 clone for IHC/IF in which the antibody works in both pH6 and pH9. we use a very standard method of boiling for 10' in the microwave in either citrate (pH6) or tris/edta buffer (pH9) and cooling down the slides for about 1h.

A possible alternative if you cannot get this antibody to work would be the D6O8R clone from CST. we currently use it for IMC with a pH6 AG, however it also works with pH9. we conjugate ourselves and use it in a 1/50 dilution.

ssivajothi
Posts: 31
Joined: Thu Jan 11, 2018 9:00 pm

Re: FoxP3 stain does not work

Post by ssivajothi » Mon Aug 19, 2019 6:25 pm

Hi kuehla,

I have had success with the 236A/E7 clone (Abcam i think). I have it custom conjugated to 165Ho and I use pH6 for retrieval in a pressure cooker for 10 mins. If a validated antibody doesn't work, I usually re-try the conjugation. If you are still having issues, try to go the secondary antibody route. Best of luck!

Santhosh

MariekeIJsselsteijn
Posts: 8
Joined: Mon Jan 15, 2018 10:52 am

Re: FoxP3 stain does not work

Post by MariekeIJsselsteijn » Tue Aug 20, 2019 6:45 am

Hi Kuehla,

another thing you can try is to do an IHC with the conjugated antibody in both pH6 and pH9. you will see some background due to the metals but you can still clearly see the specific signal from the antibody. We do this often to confirm that the conjugation hasn't changed how the antibody functions. In this way you can confirm if it is a problem with the antibody or the IMC staining method.

Marieke

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