Murine CD marker woes Re: [Histonet] any suggestions?

[Gayle Callis]

REnee,

It would be nice to know which CD marker and know exactly how he is doing 
his staining????  Including the buffer and if he uses any detergent, 
protein in buffer - serum or BSA?  A lot of murine CD IHC background woes 
are not just the secondary.   He can adsorb his secondary by simply using 
from 1 to 5% mouse serum in the diluent of secondary and in the normal 
serum block, i.e. 10% goat with 1 to 5% mouse serum for 30 minutes before 
the primary.  I have, on occasion added mouse serum to primary diluent, and 
do this routinely when diluting a biotinylated Rat antiMouse primary for a 
CD marker followed by Strepavidin-HRP.

You were correct in advising  to match the blocking serum to the host of 
the secondary.  If he wants to use a serum other than goat,  swine may be a 
better choice than rabbit.

No, frozen sections are not going to be a factor and he may not be able to 
use FFPE for the CD markers anyway, crosslinking by NBF will ruin the 
antigens to the point of NO retrieval, ever!!!

What do you mean by the stain?  Chromogen? Which one?  Hopefully he did a 
dilution panel starting with a target concentration of 10 ug/ml for 
primary, and the secondary should be good at 1:250 or approx 2 to 5 ug/ml -

Does he control the development of chromogen with a microscope?  Is he 
using HRP or AP?  Blocking? Tween 20 in buffer?  Protein additive to buffer?

How are frozen sections fixed?  Cold acetone?  Is he using an isotype 
matched IgG control for the antibody used at the same concentration?

More details would help - so I ask a lot of questions!!




  At 12:13 PM 11/9/2005, you 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'
>
>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)