Re: more info

From:Gayle Callis


It could be you are not totally quenching the endogenous and
pseudoperoxidases in the cells. A standard peroxidase block often used
hydrogen peroxide 3% with methanol, however there is a block that is total.
 Glucose Oxidase method.  I will be happy to attach that to you if you
want. It works for paraffin and in particular, frozen sections that are so
fragile/easily damaged due to minimal i.e. acetone fixation and certainly
is a help if you suspect you have peroxidases still present.   

Have you tried an isotype matched negative control Rabbit IgG or mouse IgG
1, 2a, etc.  You can match the concentration of negative control IgG
perfectly to concentration of primary antibody - a must. Normal serums are
more of a guessing game as to exact ug/ml matched exactly to your primary
antibody.  Rabbit IgG can be purchased from Jackson and mouse IgG isotype
from Pharmingen, Sigma, DAKO, etc.  

Good luck. 

At 11:47 AM 8/1/2003 -0400, you wrote:
>I'm sorry about not giving enough information.  I have had several 
>responses asking for more details.  So, I'll try again!
>I am performing IHC staining.  I am using a DAKO autostainer with 
>the LSAB detection system.  I use DAB for my chromagen.  My negative 
>mouse and negative rabbit serum slides have quite specific staining 
>in the granlocytic series of WBC's(No matter what pretreatment - 
>I apply no pretreatment, Proteinase K, or use Microwave at pH 6 and 
>10).  Microwave pretreatment is the worst, but I get the staining 
>most of the time.  Polysegmented neutrophils seem to be the main 
>culprit.  Most of the times my Docs can read through this, but they 
>are requesting my experimentation to find a "cure" for the situation,
>so they can honestly say, "no staining was seen in the negative 
>control". One of the suggestions I have received is to do an Avidin/Biotin 
>Block. I will give this a try, but if there are any other ideas out 
>there:  DON'T hold back!  THANKS AGAIN! 
>Naples Free-Net Web Mail
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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