|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.
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