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.

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.

[shankar]

Dear max,
              i am trying to get a nucler signal regarding a histone deacetylase enzyme HDAC3.The anti serum i am using is raised in rabbit by ourselves and it works excellent for immunoblots.The signal i am getting  after using this antibody is very specific for cytoplasm,but it is also expected to give a nuclear signal. I had tried all the possible methods like citrate ph 6 for 30 min, tris edta ph9 for 30 min power level 5 in microwave,tris edta ph 9 along with 37 mg trypsin/70ml pbs 37 oc for 15 min treatment also didnt  give me a nuclear signal,even i had tried pressure cooking method for 7 whistles at boiling temp but didnt tried autoclaving.i dont know where i am going wrong? in literature it was demonstrated that a nuclear signal is possible in prostate tissue.hope u can give me a clue.please can u also let me know about  a good antigen retrival protocol for acetylated histones H3 and H4 in nucleus in different tissues!i am using LSAB + kit for secondary antibody.thanking u
shankar.N.G

[shankar]

Dear all,  
      does sodium azide 0.001%, which is used as a preservant for storing antiserum ,can interfere with immunohistochemistry? if so what are the changes to be done in the procedure.

thanking you all,
shankar.N.G

[ImmunoNYC]

Generally speaking, sodium azide will definitely interfere and inhibit HRP.  However, in your case it's just in the primary stock solution and assuming you dilute your primary and do adequate washes and then your HRP labeled secondary isn't diluted in any azde containing buffer it really shouldn't be a problem.

Dear all,  
      does sodium azide 0.001%, which is used as a preservant for storing antiserum ,can interfere with immunohistochemistry? if so what are the changes to be done in the procedure.

thanking you all,
shankar.N.G

[ImmunoNYC]

The anti serum i am using is raised in rabbit by ourselves and it works excellent for immunoblots.

Hmm, I see. Perhaps your antibody isn't good for IHC then, are you the first to try it? Not all antibodies are created equal! Are you sure the antibody is specific? Is this unpurified antiserum or purified rabbit IgG? If it is unpurified this may be the issue .

The signal i am getting  after using this antibody is very specific for cytoplasm,but it is also expected to give a nuclear signal.

Are you sure thje cytoplasmic staining you are getting is real? Do you have non-immune serum from the same rabbit?