Post subject: DAKO CSA System-ImmPACT NovaRED compatibility!
I am currently doing some ophthalmology research and planing to investigate
the expression and prognostic value of Endoglin (CD 105) in uveal melanoma.
Formalin-fixed, paraffin-embedded, 10 yaers old tissue will be used.
The CSA (Dako) system was recommended to me.
But, because of the highly pigmented tumor, and obtaining better contrast in visualisation of blood vessels, I am interested if there is possibillty to use ImmPACT NovaRed instead of DAB chromogen in CSA system? Is it compatible with this system?
Your experience is welcome.
Sun May 27, 2012 4:59 pm
Joined: Oct 11, 2004
Post subject: Hello
Now, let me see.......
Sure, you can use any chromogen you want to ( Px-catalysed, of course).
Do you know if you really need to use such an expensive, highly sensitive detection ( Tyramde)???
Have you tried a conventional method, inoitially?
Such as Indirect IF ( using Alexa or Dylight dyes, for eg), or stABCpx or micropolymer kit?
ONLY if they are not sensitive enough, should you us CSA.
Of course, you can always bleach the pigment out.....
Or, use an alk-phos. based system and a red chromogen
Mon May 28, 2012 6:06 am
Joined: May 27, 2012
thank you for being interested.
I would gladly use other conventional methods and save the money, but the tissue is very fragile. That is the reason why the CSA system is suitable.
When using the CSA detection system, pretreatment of tissue with proteolytic enzymes is not recommended, and it turned out that Endoglin is very demanding.
I must say that I am an ophthalmologist and Im doing this research with my mentor who is pathologist. We do not have experience with CSA detection system and that is the reason why we should be more careful.
There is 155 paraffin-embedded tissue sections and our budget is limited
Monoclonal anti-endoglin, SN6h, may be used at a dilution of 1:2000 in the CSA detection system...which is its advantage too...
Any suggestions or advice are welcome...
Tue May 29, 2012 6:50 pm
Joined: Oct 11, 2004
Post subject: Thanks for explaining.....
If you can demonstrate CD105 without AR ( antigen retrieval) of any kind, at such a high dilution factor, that is good.
The Human Protein atlas shows some very nice endothelial cell positivity, ater Citric acid HIER ( no proteolysis).
However, they use a a different clone.
As your specimens are precious, one has to take the safest route.
SO, I asume that you will test the Ab and the CSA kit on irrelevant, posiitve control tissues before you commit to staining your precious sections?
That way you not only familiarise yourself with the detection system you also will titrate the Ab to optimise the staining ( there is NO guarantee that it will work at 1/2K for you...unless you have already tried it?).
Also, if you use Novared as your chromogen, it will give at least x2 stronger staining than a std DAB chromogen.
The CSA is pretty std: one just precipitates many, many more biotins at the site of Ab:Ag binding, as you know.
We'd be interested to know how you get on....good luck!
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