[Histonet] predicament

From:Cathy Malcontenti-Wilson

Dear All Gurus,
I have a predicament which I need to overcome.
We are doing an immuno for podoplanin on mouse tissues.
We do proteinase K for enzymatic retrieval, and Hydrogen peroxide block.
The antibody is a syrian hamster anti mouse podoplanin.
The secondary antibody is a rabbit anti syrian hamster IgG.
We then use the DAKO envision plus kit (for rabbit primaries). In this kit, 
the tertiary antibody is rabbit ant goat IgG HRP conjugated.
Problem: heaps of background staining on the negative and positive.
After much trouble shooting, we have found that if we omit only the 
secondary antibody, we get no backgound. Does this mean that the secondary 
antibody (rabbit anti syrian hamster IgGs) is sticking non specifically to 
the tissue and causing the background?
We have also tried using normal rabbit serum or FCS as a normal serum block 
but this makes very little difference.
Also some serum and detergent in the antibody diluents makes very little 
Other controls: if we omit only the primary: we get background staining
If we omit all antibodies, we get no staining (so its not the endogenous 
peroxidases giving the background)
If we omit the tertiary antibody only, we get no background staining.
If we omit the proteinase K we get background staining.
Does anyone have any ideas about how we can reduce this large amount of 
background staining? Or perhaps we are doing something wrong.
Also, does anyone have a good list for the types of controls to do when 
troubleshooting immuno staining problems? I would love to use this as a 
resource in our lab.

Thanks very much.

Histonet mailing list

<< Previous Message | Next Message >>