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Author Topic: secondary Ab background problems  (Read 3598 times)

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Offline adamo

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secondary Ab background problems
« on: February 15, 2007, 12:12:03 PM »
I am using the Elite Vectastain mouse IgG kit to do IHC on cryosectioned (12um) mouse embryos.  I have no background problems with embryos ages E8.5 to E11.5 but when the embryos are older (E15.5 and E17.5) the secondary antibody binds to EVERYTHING and I loose specificity.  I tried titrating in NaCl as per the Vector troubleshooting guide (this worked great for me in the younger embryos) and even titrating down my secondary but with no success.  I tried detergents in the antibody diluents with the younger embryos but that did not work so I assumed it would not work with the older embryos as well.  Has naybody experienced the same thing?  Can somebody offer any suggestions as to how I can bring down the background from the secondary antibody and still preserve my primary antibody specificity?

secondary Ab background problems
« on: February 15, 2007, 12:12:03 PM »

Offline ImmunoNYC

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Re: secondary Ab background problems
« Reply #1 on: February 16, 2007, 12:13:04 AM »
Isn't the Elite Vectastain Mouse IgG kit for staining mouse monoclonal antibodies on other tissue besides mouse? I suspect your secondary is cross reacting with endogenous IgG in your tissue? What is the species of your primary? I think you are using the wrong kit perhaps?

My suggestion is to scrap the Vector kits and build your own protocol. It's easy and I can help if you give me more info on your antibody (host it's made in is a good place to start). Otherwise if you are staining with a mouse Ab on mouse embryos you need to switch to a Mouse-On-Mouse kit (MOM or ARK kits) for example.

Offline adamo

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Re: secondary Ab background problems
« Reply #2 on: February 16, 2007, 10:19:40 AM »
NYC

Thanks for the heads up.  I never considered that and I am even a bit embnarassed by that now that I think about how obvious it should have been.  I guess I was lucky to have it work with the younger embryos and so it confused me why the older embryos were giving me such a big problem.  Unfortunately most of the important primaries I am using are mouse so a MOM kit would be best here.  I believe Vector makes one, do you have any suggestions as to where else I could find one that works well?

Offline ImmunoNYC

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Re: secondary Ab background problems
« Reply #3 on: February 19, 2007, 01:43:49 PM »
I like the kit from DAKO, the Animal Research Kit (ARK), my lab also uses MOM kit with much success. What are your antigens you are staining for? It's unfortunate that you are limited to doing mouse on mouse, are there really no other good Abs for IHC for those antigens? If you tell me what they are perhaps I will have some suggestions.

Offline wszyy

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Re: secondary Ab background problems
« Reply #4 on: February 28, 2007, 01:04:53 AM »
same problem as me. I am using mouse on mouse Ab. My target signal is fine, but the background is too high and the picture is so ugly. I did not use any kit. how can i improve it?

Re: secondary Ab background problems
« Reply #4 on: February 28, 2007, 01:04:53 AM »