RE: [Histonet] troubles with CDK4
Try using your serum free protein block before the secondary reagent as well
as before the primary.
In my experience most non specific staining is from one or more of 5 things:
1. The secondary or labeled polymer is binding non specifically to the
tissue (this can
be alleviated by using a really good f(ab)fragmented secondary which has
to the species of your primary antibody).
2. The endogenous peroxidase has not been quenched.
3. There is endogenous biotin left and you are using an AB detection
4. You have over treated by HIER or EIER (this often shows up as unexpected
5. What is thought to be non-specific staining is actually specific binding
I am sure there are many other causes for non specific staining, but in my
experience these five are most common.
Did you get the same BG with LSAB detection that you got with labeled
polymer? If so, then it is not the endogenous biotin causing your problem.
Also, to really find out, run your slides adding one step at a time to see
where you get non specific staining. Forinstance, stain a set of slides
leaving out the primary antibody for all, don't block with h202 and do the
DAB to see what it looks like with out blocking, do the block for endogenous
peroxidase and then go directly to DAB leaving out the secondary and primary
steps and if you are still getting staining that means your peroxidase block
is not working properly, keep adding the detection reagents without the
primary antibody until you get staining. If you don't ever get staining
until you add the primary antibody then you can assume that it is specific
staining related to that antibody. Sometimes we get undesired "specific"
staining, which we often call "non specific".
Patsy Ruegg, HT(ASCP)QIHC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
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[mailto:email@example.com] On Behalf Of
Sent: Tuesday, February 07, 2006 7:44 AM
Subject: [Histonet] troubles with CDK4
I am trying to do IHC with rabbit anti human CDK4 (D-22) from Santa Cruz on
salivary gland tissue. I am having background problems which is leading to
difficulty in figuring out what is positive and what is negative. I have
used LSAB2 kit from Dako, Rabbit Envision Polymer from Dako and the Rabbit
ABC Elite kit from Vector. I do a 3% hydrogen peroxide treatment for 20
minutes and do antigen retrieval with 10mM Citric Buffer pH 6.0 for 45
minutes in a steamer and a 20 minute cool down. I do a blocking step and
have used either serum free or goat serum for a block.
I already checked with the company about my protocol and it is the same as
theirs. I tried doing it on skin which is reccommended as a control but
still have background problems.
Senior Research Assistant
MD Anderson Cancer Research Center
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