[Histonet] any suggestions?

[Andrea T. Hooper]

Dear Renee,

It is going to be essential that his secondary antibody be 
cross-adsorbed against mouse even though his primary is anti-F(ab')2. 
When I switched over the non-cross reacting secondaries my whole 
quality of staining mouse tissue improved a 1000%.

In addition, I suggest he run a panel of controls if he hasn't 
already including:

(1) no primary but with secondary and strep-HRP  (non-specific 
secondary binding control)
(2) isotype control with secondary and strep-HRP  (primary FcR 
interaction and non-specific binding control)
(3) Only strep-HRP (biotin control)
(4) Only DAB (peroxidase control)

Cheers,
Andrea




At 1:13 PM -0600 11/9/05, Till, Renee wrote:
>Another tech who does not have much experience with histology came to me
>with questions about his immunos. They are doing IHC with various cd
>markers on frozen sections of mouse aorta. He has encountered
>particularly strong background(or so he's been told, he thinks it is
>actual staining) with one of the antibodies that was made in rat. He is
>using a goat anti-rat F(ab')2 from Jackson as the secondary. It is not
>absorbed against mouse. I have asked all about his dilutions and
>incubations times, but he doesn't seem to think that is the problem. I
>gave him an avidin/biotin block to try and see if that helps. Any other
>ideas? I am not familiar with cd markers myself. The only problem I
>could find just in talking to him was that he was blocking with rabbit
>serum? I told him you normally match your block with the host of the
>secondary, but would that make that big a difference as far as
>background is concerned?  Would the fact he is using frozen sections
>have anything to do with it? Or could it just be the stain? I know they
>are doing cd54, but I'm not sure if this is the one he is having a
>problem with.
>
>I know this is not much informations, but I would still appreciate any
>input
>
>Thanks,
>
>Renee'
>

--