I am trying IF in the 4%PFA fixed paraffin embedded mouse ovarium tissue. I always got high backgrounds (non-specific binding). I applied sodiumcitrate buffer (pH: 6.0) and Tris-Edta buffer (pH: 9.0) for the 20 min in microwave (max power and 600 wat). Block endogenous peroxidases with 3% H2O2 at RT for 30 min (in PBS). I am blocking the tissue with 3% goat serum +1% BSA + 0,1% TritonX-100 + 0,05% Tween-20 in PBS at RT for 30 min. I would appreciate any suggestions on how I could get rid of these non-specific binding.
HI Ozlem,
You started in a right direction, If any of those steps did not resolve your issue. Try doing skipped ( No Primary ab) stain. If you don't get high back ground there then most likely it's your primary antibody's concentration that causing it to cross react. Try doing some serial dilutions of antibody and see if that help.
in a case where you get high background in skipped stain, Try diluting your secondary antibody.
Good Luck!
I understand, that you perform immunofluorescence and you don't perfom chromogenic immunohistochemistry. Peroxidase-block makes only sense, if you develope your signal with peroxidase-chromogene-detectionsystems (DAB) for light microscopy.
Make a search in researchgate for "quenching" and/or "autofluorescence". There are several questions and answers regarding this issue.
Autofluorescence on fixed specimens is an old and recurrent problem. Binding of formaldeyhde to tissue-proteins produce some kind of fluorochromic compounds, that give high background. Therefore unfixed tissue and frozen sections are prefered. Additionally some endogenous proteins or pigments for themselves give autofluorescence.
(Autofluorescence is actually a wrong term, because this would mean, that the compounds "shine" without excitation.)
Hello, you can, like Gudrun say, verify the autoflorescence of your tissue. But what maybe happing is a block overload. Try to use one block a time, and for second try two kind os blocks in a row. And in the end if is need you can try without block. Like i did in my monkey liver staining and was perfect, but that was in a cryogenic tissue, not a paraffin embedded one. But in the pig liver, that was paraffin embedded, i use only goat serum and tween-20. I wish you a god experiment, non-specific staining is very annoying in IF! God luck!!
You shouldn't need to do an endogenous peroxidase block block if you are using a fluorescent tag. It would be needed for a peroxidase-chromogene-detectionsystems (DAB) for light microscopy. IF on paraffin sections is problematic because of the processing, but is possible with a confocal microscope.
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