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i'm wondering... how do you validate your IHC? how do you know where to look at. how do you know your signal is sensitive enough?
thanx,
marcel
Sat Nov 01, 2008 9:20 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hi Marcel...
I now only carry out manual IHC in a research lab but your validation concerns are relevant to any Immunohistochemical assay.
Please post this in the other IHC forum, also....
There are many , many levels of validation.
The main one is Peer-group review, for experimental Abs.
For validated Ab reagents, for Clinical diagnostic use, each Clinical lab should be part of an external Quality assessment Scheme that regularly assesses each labs' immunostainings.
On this site, we attempt to show images of Immunostainings that are validated.....so, for example, if you want to check out if your anti CD3 is working well, just go into the image gallery and look at submitted images.
After looking at the images you may have further Qs to ask....please ask more, more in the appropriate Forum.
Re external IHC quality assessments: in UK we have an established, very well respected External Quality Assessment Scheme.
This panel is made up of experts in the field who regulary send out sections to labs, asking them to immunostain them for specific Ags.
Once the immunostained sections are returned, they are assessed and the results are posted back. So, any underperforming labs will know that their Immunostaining does not come up to National Stds.
The Panel will always offer assistance so that any underperforming lab can quickly be helped.
I hope that others here will give better answers, Marcel.
Please do not forget Histonet! Another important site to post such an important question!
Best wishes
carl
Eg: whenever anyone uses an anti CDx,y.... Ab, it must be understood that "CD" means "cluster of Differentiation"...that means that a whole load of International experts get together to decide that any particular Ab is valid for a particular protein!
Remember, proteins are BIG.....each Ab that is said to be against any particular protein will only be raised against specific peptide sequences: one sequence, if it is a monoclonal Ab, many more, potentially, if it is a polyclonal!
Also bear in mind that any monoclonal/polyclonal may/not be pwax reactive and may be only good for frozen sections/cells or FACS analysis.
So, validation of any Ab is quite a complicated process..
Too complicated for a simple guy like me....loadsa Experts out there.
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