[Histonet] RE: Neg controls

[Malam Jacqueline]

Check you are using the correct sera for your negative controls - should be
the "Ig fraction" sera (the "whole sera" is used for the blocking stage). 
Your sera could be too low a dilution - you need to dilute it to the same
protein concentration as the diluted primary. There is a formula:
If dilution of primary=A
and total protein concentration of primary =B
And total protein concentration of neg. sera=C
you divide A by B and multiply by C
Then you just treat the slide to the same ag. retrieval process etc as the
primary.
You do end up with a lot of neg. control slides (no retrieval, enzyme,
pressure cook, microwave, pressure cook and enzyme) both for polys and
monos. We used to do the lot but now we are fully automated and there is
only one poly and mono sera available on their commercial list so, based on
the theory that if it's ok with them to stick with that, when we make our
own antisera preps, we dilute both rabbit and mouse Ig sera 1/1,000. It's an
arbitrary figure based on the recommended dilution for mouse sera from Dako
years ago. If there are any problems (and there haven't been yet), we could
always repeat with a "corrected for total protein concentration" dilution. 
Good luck!
Jacqui



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