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Author Topic: How to reduce non specific staining of your primary antibody?  (Read 18953 times)

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Offline JB22

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How to reduce non specific staining of your primary antibody?
« on: February 03, 2011, 04:11:08 AM »
Hi,

I am trying to setup a staining for CRE and Beta-gal using DAB on mouse embryo and hart tissue sections embedded in paraffin. With both antibodies I get a lot of non specific staining. This happens for all the dilutions of primary antibody that I have tested and going any lower than the strongest dilution I have tested will probably reduce the specific staining to much.  The non specific staining seems to be coming from my primary antibody, a rabbit anti mouse, and not from my secondary, a swine anti rabbit, because the negative control in which I use 1% BSA/PBS instead of primary shows no staining.

From my limited understanding of immunohistochemistry I know that to prevent non specific staining of your secondary you can use a blocking step with things like serum and BSA. But what can you do to prevent non specific staining coming form your primary?

Thanks in advance.

How to reduce non specific staining of your primary antibody?
« on: February 03, 2011, 04:11:08 AM »

Offline funk.106

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Re: How to reduce non specific staining of your primary antibody?
« Reply #1 on: February 03, 2011, 09:28:18 AM »
Blocking in BSA or serum will prevent non-specific binding from your primary in addition to non-specific binding from your secondary. Personally, I use the serum that my secondary was raised in. So if yours is swine anti-rabbit, use 5% swine serum diluted in PBST. Also dilute your antibodies in say, 2.5% swine serum diluted in PBST. Also be sure that you are thoroughly washing after the primary antibody incubation step.

Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #2 on: February 03, 2011, 09:58:16 AM »
As you are using DAB, I am supposing you have a HRP-conjugated secondary.  Are you doing an endogenous peroxidase block as part of your protocol?

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #3 on: February 04, 2011, 03:31:25 AM »
Interesting, I never knew that blocking has effect on your primary. At the moment I block in 1% BSA for 1h. So I think a test with different blocking methods is a good next step.

Yes I block endogenous peroxidase by incubating for 20 min in 0,3% H2O2 in PBS right after re hydrating and before antigen retrieval. Sins my negative controle has no staining i would not expect that the background comes from endogenous peroxide or am i missing something?

I use PBS in the washing steps, 3x5 min while shaking lightly, but I have seen a lot of protocols who use PBS-Tween instead. Could washing in PBS-Tween help to reduce background? Are there any other washing methods that might help?

At the moment I use boiling for 10 min in Citrate buffer pH 6 for antigen retrieval, could using a different antigen retrieval method help to reduce background from my primary antibody?
« Last Edit: February 04, 2011, 03:36:25 AM by JB22 »

Offline funk.106

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Re: How to reduce non specific staining of your primary antibody?
« Reply #4 on: February 04, 2011, 09:17:20 AM »
I use PBS with tween in my washes....the tween will help wash away some of the weaker non-specific binding better than PBS alone. I think you are right that the background is not coming from endogenous peroxidase since you're not seeing it in negative control. As far as my experience goes, I don't think that the method of antigen retrieval will affect background a whole lot unless you over retrieve---but I microwave for 20 minutes in citrate, so 10 minutes doesn't seem like it would cause that. I would definitely try blocking with serum if I were you and add the tween to your washes and see how that helps. If you don't have swine serum, then just make sure that its not the serum of what your antibody detects (ie, if it's swine anti rabbit, don't use rabbit serum to block).

Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #5 on: February 04, 2011, 11:51:13 AM »
Yes I block endogenous peroxidase by incubating for 20 min in 0,3% H2O2 in PBS right after re hydrating and before antigen retrieval. Sins my negative controle has no staining i would not expect that the background comes from endogenous peroxide or am i missing something?

I block peroxidases after antigen retrieval as you can expose additional activity with retrieval.  

What are you using as a negative control?
« Last Edit: February 04, 2011, 12:00:39 PM by MT Scientist »

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #6 on: February 04, 2011, 12:32:40 PM »
Thanks for the tips so far.

My negative control is on a slide from the same embryo/hart but I use 1% BSA/PBS instead of primary antibody.



Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #7 on: February 04, 2011, 02:49:44 PM »
My negative control is on a slide from the same embryo/hart but I use 1% BSA/PBS instead of primary antibody.

I hate to assume anything...
Is the control slide undergoing the retrieval?
Are you using a polyclonal or a monoclonal?
What dilutions of primary and secondary have you tested?

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #8 on: February 06, 2011, 11:17:23 AM »
Yes the controls undergo retrieval. For the negative control I follow the same protocols as for the other slides with the adjustment that I use no primary antibody.

The primary's are polyclonal. The secondary I do not know by hart, will check tomorrow at work.

The dilutions that I have checked are for the Cre 1/300,  1/500, 1/1000, 1/5000, 1/10000.
For Beta-gal 1/500, 1/1000, 1/2000, 1/4000.

At this point my plan is going to be to use serum for blocking, use PBS-Tween for washing and I will perform the endogenous peroxidase block after the antigen retrieval. I am also going to use an extra negative control to make sure that what I think is positive staining is indeed positive. For this I am planning to stain a section from a wild type mouse embryo.
« Last Edit: February 06, 2011, 11:21:07 AM by JB22 »

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #9 on: February 07, 2011, 10:59:26 AM »
I was correct, my secondary antibody is also a polyclonal.

Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #10 on: February 07, 2011, 11:44:12 AM »
Have you tried titrating your secondary?  Too much secondary can also cause background problems....

A matrix test with multiple primary & secondary dilutions will get you in the ballpark and then focus on titrating around the dilutions that give you the best signal to noise ratio.

Offline JB22

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Re: How to reduce non specific staining of your primary antibody?
« Reply #11 on: February 14, 2011, 07:36:22 AM »
No I never change the concentration of my secondary. The college who taught me how to stain told me that it is preferred to have the same concentration secondary for all primaries. I asked the reason behind this and he told me that it is more practical if you do a large amount of slides simultaneous with different antibody's to have the same concentration for your secondary.

Last week I tried several of the suggestions I got so fare for my Cre antibody. In short I did the following:

-   Rehydrate
-   Antigen retrieval, boiling 10 min in citrate buffer pH 6
-   Endogenous peroxidase block
-   Draw circles with Pap pen.
-   Wash 1x in PBST 0,05%
-   Block 1h RT, 10% serum PBST (0,05% Tween)
-   Cre o/n in 10% serum PBST (0,05% Tween)
-   Wash in PBST (0,05% Tween)
-   Secondary 30 min RT
-   Wash PBST
-   ABC in 10% serum PBST, 30 min RT
-   Wash PBST
-    Wash PBS
-   DAB
-   Hematoxyline 30 sec, tap water 10 min.
-   Dehydrated and cover.

For Cre antibody I used 1/500, 1/1000 and 1/2000, for my secondary I tried 1/200 (our standard dilution), 1/500 and 1/1000. I used an extra negative control by staining a slide from a WT mouse embryo which should not have any Cre

Unfortunately the changes have not solved my background problem. My negative controls were I used 10% serum PBST instead of primary are not stained. But my extra negative control stains brown. In the links below you can find 2 pictures at different magnifications, a 2,5x and a 25X, from my extra negative control (Cre 1/1000 and secondary antibody 1/200).

http://i1178.photobucket.com/albums/x370/s999359/Extranegativecontrole25X-1.jpg

http://i1178.photobucket.com/albums/x370/s999359/Extranegativecontrole25X.jpg


Any suggestions on how I should proceed?


Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #12 on: February 14, 2011, 09:52:37 AM »
No I never change the concentration of my secondary. The college who taught me how to stain told me that it is preferred to have the same concentration secondary for all primaries. I asked the reason behind this and he told me that it is more practical if you do a large amount of slides simultaneous with different antibody's to have the same concentration for your secondary.


No offense to your colleague, but that logic only gets you into trouble.  So it takes 30-45 seconds more to make a different dilution of secondary...Why should that matter if your data is better and cleaner?  Try diluting your secondary.....

« Last Edit: February 14, 2011, 09:57:06 AM by MT Scientist »

Offline MT Scientist

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Re: How to reduce non specific staining of your primary antibody?
« Reply #13 on: February 14, 2011, 10:30:05 AM »
I am assuming ABC is an avidin-biotin amplification.  If so, I am not seeing a blocking step for endogenous biotin.  This could be causing your background issues....


« Last Edit: February 14, 2011, 12:35:23 PM by MT Scientist »

Offline sabo

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Re: How to reduce non specific staining of your primary antibody?
« Reply #14 on: February 18, 2011, 02:26:51 AM »
Hi,
I am doing IHC for Carbonic anhydrase IX, which is rabbit polyclonal. My secondary is polymeric HRP-linker antibody conjugates. Even after performing all the blocking steps properly, I am still getting background staining in my negative control. So I assume its because of endogenous peroxidase. In antigen retrieval, am using pressure cooker method for Citrate Buffer(pH-6;10 mins). And for washing only TBS.

Is it better to use PBS/ PBS with Tween for better washing? what else should I do to reduce the background stain?

And I have a doubt about the interaction which takes place between the non-specific sites and the block, containing the serum. I could not find the answer in Internet. Can you please tell me something about this interaction. How does 'only' the non-specific sites recognize the blocking agent?

Re: How to reduce non specific staining of your primary antibody?
« Reply #14 on: February 18, 2011, 02:26:51 AM »