Antibody-Based Technologies > Antibody H

Histone IHC - HELP! urgent

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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.


--- Quote from: "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
--- End quote ---

ImmunoNYC:

--- Quote from: "shankar" ---The anti serum i am using is raised in rabbit by ourselves and it works excellent for immunoblots.
--- End quote ---

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 .


--- Quote from: "shankar" ---The signal i am getting  after using this antibody is very specific for cytoplasm,but it is also expected to give a nuclear signal.
--- End quote ---

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

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