Re: [Histonet] Negative controls

From:"Connie McManus"

I missed what antibody you're talking about and what detection system you're using, but
in general, this is how I was taught to treat assays that use a neg ab ctrl:

The negative antibody must be run with the same protocol as the pos antibody and is
diluted to the same concentration.

Treat positive and negative ctrl tissues with pos and neg antibodies (use the same
tissue ctrls for both abs, each ctrl slide has pos and neg tissue.  I embed the pos and
neg tissues in the same block and orient the neg ctrl to the top of the slide so I would
always know which tissue was which).

If you are getting staining with your negative antibody, it could be background
staining.  This may be alleviated by...
1- check the blocking:   a/b block, h202 or general protein blocking (if you're using
Alk Phos, you may need to treat the sections with levamisole - h202 is not needed).
2- antigent retrieval timing needs to be revised (if you are using a/r) -- you may need
to find a balance between the timing that makes a good pos rxn in the pos ab and no
background in the neg ab.
3 - Be sure the detection system is compatible with your tissues -- i.e. don't use alk
phos on tissues rich in endogenous alk phos and the same with peroxidase.

The other possibility is that the neg ab ctrl may be contaminated.  To avoid this, I
always aliquotted small volumes into cryo vials and stored them as per manufacturer's

Each new lot of antibody is qc'd along with titering to assure the working dilution is
acceptable.  Neg ab ctrls are run alongside the pos ab at this time.  Records of the
results are filed.

I hope I covered all the bases here.  I may have forgotten a thing or two, but I'm sure
if I did leave something out, there are those who will  bring it up.  I think, in a
nutshell, the basic rule of thumb is to treat the neg ab ctrl exactly the same as the
pos ab.

hope this has been helpful.
Connie McManus
Mt Ogden Scientific Services -Providing full service electron microscopy for you
950 W Kershaw, Suite E
Ogden UT  84401
toll-free: 877/311-6677
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Amy Johnson said:
> Our laboratory has been going thru the new Anatomic Pathology Checklist for
> CAP and we came across
>  the Question "Are appropriate negative controls used?"  We use to run one
> protocal for the negative
> mouse antibody and one protocol fro the negative rabbit antibody which was:
> 	no antigen retrieval
> 	no enzyme
> 	antibody incubation was only 2 minutes
> 	no amplification, no A/B block
> 	counterstain and post counterstain
> Now since we reviewed the CAP checklist we found out we were wrong, that we
> need to run the negative
> protocol the same as the primary antibody protocol.  We have tried this and
> been having problems.  We are getting
> positive staining in our negative controls even though there is no primary
> antibody being applied.  How is this happening?
> How can this be solved?
> Thanks in advance for the help,
> Amy Johnson
> Associates in Pathology
> Wausau   WI
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