Antibody-Based Technologies > Antibody H
Histone IHC - HELP! urgent
shankar:
Dear all,
i am badly need in help regarding the immunohistochemistry for a histone modifying enzyme which was supposed to be present in the nucleus and cytoplasm of mammalian cells depending on the status of the cell.while i perform the IHC against to this antigen using a ployclonal antibody serum at 1:50. 1:100 dilutions, i am able to get the cytoplasmic signal which is very specific, but not the nuclear signal.
i had tried all the antigen tetrival mentods like citrate ph6 for 30 min at 500w in a microwave.got a good cytoplasmic signal but not nuclear signal.
i had tried trypsin at 35mg/70 ml of pbs at 37 o c for 15 min in a microwave and then leaving it at 30 min at RT.got a god cytoplasimic signal but not nuclear.
i had tried pressure cocker method for 7 whistles at boiling temp in tris edta ph9.got cytoplasmic signal but not nuclear.signal is comparatively weak relative to trypsin method.
it is published that this protein resides in nucleus in certain tissues and IHC is very clearly demonstrated by alpplying the method of pressurised microwave which unfortunately i cannot work on.
i strongly belive that the tissue i am working on will also show the nuclear signal.
PLEASE ANY ONE CAN KINDLY SUGEST ME THE ANTIGEN RETRIVAL TECHNIQUE FOR GETTING THE NUCLEAR SIGNAL!!!!!!
thanking you in advance.
richard03:
I suggest you try EDTA buffer antigen retrieval method. If it did not work. You may consider to replace your primary ab with a different vender or clone.
Richard
shankar:
Dear richard,
thank you for the suggestion.i will try the EDTA Ph8 method now.can u please suggest me the best suggested maximum time for the antigen retrival in a microwave for nuclear antigens, and should i try a double antigen retrival using trypsinisation.if so can u suggest me the time and concentration(trypsin) for the antigen retrival which works best for the nuclear antigens.
shankar.N.G
richard03:
Dear Shankar,
I like EDTA more than other antigen retrieval methods for nuclear antigens. I don't see the need for combining with enzyme digestion to reveal nuclear antigens. The optimal retrieval time varies depending on fixation, tissue type, etc. but 20 minutes is commonly used. Less than 10 minutes retrieval is usually insufficient.
Richard
ImmunoNYC:
Is your antibody the same clone, from the same vendor? This is most often the problem. Where is your antibody from? I understand Upstate Biotech sells the best ones for histone modifications. Which modification is it? I have experience with a few of them and perhaps can help more.
--- Quote from: "shankar" ---Dear all,
it is published that this protein resides in nucleus in certain tissues and IHC is very clearly demonstrated by alpplying the method of pressurised microwave which unfortunately i cannot work on.
i strongly belive that the tissue i am working on will also show the nuclear signal.
--- End quote ---
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