I am interested in studying neuroendocrine network by immunohistochemistry in formalin fixed bone marrow biopsy samples. Standard techniques of immunohistochemistry commonly used to study neural/endocrine tissues have proved disappointing. I think the problem is due to loss of antigen epitopes (or failure to retrieve them) as a result of harsh treatment the samples go through (decalcification). Are there any modified protocols that could be adapted for this purpose?
Hi Muttuswamy, I'm sorry to hear you are having troubles.
Is the problem low signal or high background (or both)?
I recently had to do some pretty tough IHC on lung tissue that had been "well preserved" so I can sympathise! I would suggest that you try a variety of AR (antigen retrieval techniques) for each antibody (for example swap the Citrate pH6 to a Tris pH 9.0 AR step). You might also want to try trypsin AR (0.25% trypsin 15 min) in place or on top of the Citrate/TEB AR step (I found this helped for several of my antibodies although it can loose the sections on the slide so be gentle).
Unfortunately for an new ab for IHC I sometimes have to a) titre antibody b) try various blocks (1%BSA-5%BSA, 1%, 10% 100% FBS) c) test against various AR techniques. This can all be a lot of work - even then there is no guarantee that a particular antibody will work (depending largely on where the epitope is in the target protein).
One of the best "quick fix" methods I found was to include a "penetration enhancer" of 0.5% Triton X-100 alongside the primary incubation (overnight 4oC)
"The antibody buffer was composed of PBS 0.1% Tween, 1% BSA and 0.5% Triton X-100 (as an enhancer) and 0.5 mg/ml DAPI".
I was surprised how well this worked (as I would normally permeabilise in only 0.1% so I would have never thought to use 0,.5%!). this tip was from IHC world (on-line)
Also do not include any BSA in with your secondary (as this can reduce signal).
Finally (and I am sure you have already) check that the antibody is suitable for IHC (I always check the antibody catalogue number on-line before buying to see if it has been used previously to give good results in the tissue under investigation).
When all else has failed you may have to buy another antibody (raised against a different epitope). If so go for a polyclonal if possible as this will maximise the chance that at least one of the epitopes might be unmasked.
Sorry I can't be more help, but have some consolation from the fact that IHC is probably the toughest antibody technique around.....
Regards,
Gary
Thank you very much for your very useful suggestions. We are going to try these out.
Kind regards
Siva
BTW where are you based at?
LGC at the Teddington site - I mainly do Cell Biology but occasionally I dabble in IHC as well qPCR/dPCR etc.
All good fun!
I just noticed that an error occurred when I copied and pasted part of my IHC protocol:
The primary antibody incubation buffer contains 0.5 ug/ml Dapi (not 0.5 mg/ml).
I lost the Greek formatting during copy:paste: sorry about that.
Not at all. I do this all the time. Thank you again for your helpful suggestions
Kind regards
Siva
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