Author Topic: Antigen retrieval buffers  (Read 19376 times)

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Offline ASluo

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Antigen retrieval buffers
« on: April 27, 2008, 01:52:35 AM »
Hi guys, there are many different types of antigen retrieval buffers to unmask antigen epitodes of formalin fixed, paraffin embedded tissues such as Tris-EDTA, citrate, sodium citrate and EDTA buffers at different cocentration. Apart from personal preferences, what are the difference? Also some recipes include the addition of 0.05% Tween 20, what's the benefit? reason?

Many thanks,
Annie

Antigen retrieval buffers
« on: April 27, 2008, 01:52:35 AM »

Offline excalibur

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Re: Antigen retrieval buffers
« Reply #1 on: April 28, 2008, 01:05:23 PM »
The differences in the different solutions is pH. Citrate buffers are scidic, while EDTA, etc. are basic. The Tween is a surfactant ( mild detergent) and helps with permealization.

Different antibodies may require an antigen retrieval solution of a certain pH to work.

Also an enzymatic antigen retrieval may be what is needed for some antibodies to work such as trypsin, pronase, etc.
Paula K. Pierce, HTL(ASCP)HT
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Offline ASluo

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Re: Antigen retrieval buffers
« Reply #2 on: April 29, 2008, 09:37:51 PM »
Thanks for the explanation! I used Tris-EDTA with 0.05% Tween 20 for antigen retrieval for FFPE mouse spleen. Unfortunately, the test samples were all clean as in absolutely no staining at all! I stained for CD3 (1:200), F4/80 (1:400) and FoxP3 (1:100), which according to the antibody specsheet that these antibodies should work in FFPE tissues. I'm thinking it might be due to the antigen retrieval buffer or the concentration of the antibodies? What are your thoughts?

Thanks,
Annie

Offline richard03

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Re: Antigen retrieval buffers
« Reply #3 on: April 29, 2008, 11:24:55 PM »
CD3 should work well with Tris-EDTA pH 9.0 but it may also depend on the antibody source.

Most F4/80 antibodies work well with either citrate buffer pH 6 or proteinase K method so the Tris-EDTA may not be the best choice for F4/80 antibody.

Offline ASluo

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Re: Antigen retrieval buffers
« Reply #4 on: April 30, 2008, 12:13:07 AM »
I
CD3 should work well with Tris-EDTA pH 9.0 but it may also depend on the antibody source.

Most F4/80 antibodies work well with either citrate buffer pH 6 or proteinase K method so the Tris-EDTA may not be the best choice for F4/80 antibody.

Is a polyclonal rabbit anti-human CD3 from DAKO. I was told the antibody will work in mouse tissues.

Offline CanuckPhD

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Re: Antigen retrieval buffers
« Reply #5 on: April 30, 2008, 05:19:36 AM »
The antibody may work in mouse tissue but there many conditions that will affect its ability to stain. These include the amount of time and fixative used, how long and at what temp you did antigen retrieval. All of these variables will affect the staining of your antibody. Plus all of the other IHC conditions (quenching, blocking, secondary etc.). Therefore you should try different dilutions of your primary and some different times of antigen retrieval ( I usually try 5, 10 and 20 minutes at 98 degrees). In my lab we usually try different retrieval solutions to test which is best. It is sometimes a long process but usually leads to better results in the end.

The data sheet should only be taken as a guide for the dilutions. I always go at least 1 dilution below and 2 above to check for staining. Antibodies are expensive and if you can get good results at a higher dilution then all the better.

Good luck

Offline ASluo

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Re: Antigen retrieval buffers
« Reply #6 on: May 04, 2008, 01:54:32 AM »
I tried the citrate buffer pH 6 and still no staining at all! I stained for CD3 (1:200), F4/80 (1:400) and FoxP3 (1:100), overnight incubation at 4oC. Tissue is mouse spleen. I'm going to increase the dilution, incubate at RT for 1hr. Any suggestions?

THanks,
Annie

Offline biotech72

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Re: Antigen retrieval buffers
« Reply #7 on: May 13, 2008, 12:48:18 PM »
Thanks for the explanation! I used Tris-EDTA with 0.05% Tween 20 for antigen retrieval for FFPE mouse spleen. Unfortunately, the test samples were all clean as in absolutely no staining at all! I stained for CD3 (1:200), F4/80 (1:400) and FoxP3 (1:100), which according to the antibody specsheet that these antibodies should work in FFPE tissues. I'm thinking it might be due to the antigen retrieval buffer or the concentration of the antibodies? What are your thoughts?

Thanks,
Annie

Hi Annie,

as far as I know the F4/80 antibody works well after antigen unmasking with Sodium citrate and it should be used more concentrated (e.g 1:50). However the antigen retrieval protocol depends on the F4/80 clone you are using and it should be indicated in the antibody data sheet.
In my hands this antibody worked on mouse spleen although spleen has a lot of endogenous biotin which stains the red pulp, exactly like the F4/80 antibody.
As last option, I suggest you try this antibody on cryosections. Normally, most antibodyes that do not work on paraffin embedded sections are supposed to work well on crysections.

Hope my recommendations could be helpful.

Good luck

Offline ASluo

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Re: Antigen retrieval buffers
« Reply #8 on: May 13, 2008, 11:15:57 PM »
Hi biotech72,

Thanks for your recommendation.

I have tried on frozen sections (normal mouse spleen and colon) and it stained beautifully with the mentioned dilutions. However, I used the normal tissues for optimisation and the real tissues are mouse colitis tissues. With the colitis tissues, there was very bad background and the morphology was absolutely terrible (please go to the gallery and have a look if you are interested). That's why we decided to use paraffin sections and see if we could get the staining to show up (especially foxp3 and F4/80).

I've tried citrate ph 6. and tri-EDTA ph 9. (microwave) and didn't work at a 1:50 dilution. I've been getting feedback about AR with proteinase K for F4/80 and will give it ago once it arrives. Annie ;)

Re: Antigen retrieval buffers
« Reply #8 on: May 13, 2008, 11:15:57 PM »