I have found our procedure to be unreliable often and cannot explain why. Does anyone have a procedure that is consistant most of the time. I do not care which antibody I need to use to test the procedure. Thanks in advance.
Dear Jackie, wouldn't this be a thoughtful post in the Topic "Electron Microscopy" ==> http://www.researchgate.net/topic/Electron_Microscopy/ ?
Unfortunately I am not able to help you out with a "100% recipe/procedure" but/and there are a lot other sophisticated people and colleagues out there... I guess.
Best wishes and good luck,
Wolfgang MUSS, Salzburg, Austria
Thanks for your suggestion. I am not sure why it did not post on that site. I appreciate your feedback. Jackie Williams, St. Jude Children's Research Hospital, Memphis, TN, USA
Dear Jackie, is your question still unanswered? We're doing pre-embedding immunoEM routinely with insoluble nuclear proteins, so we do cell permeabilization and our stainings work perfect (Nanogold+Ag-ehancement). Is this what you need? I can provide a detailed protocol
Preembedding is good if you are looking for surface antigens. For internal antigens, you need permeabilization properly that usually is not very good for structures. You can follow this flow, fixation-thick sections-immunostaining-osmification-plastic embedding-sectionig TEM.Do immunostaining with permeabilzation and go ahead with embedding procedures using any resin (you are confortable with) do sectioning, staining (only lead citrate is good enough) and scanning as usual.
Quite correct, Shashi! To circumvent these limitations we employ in vivo immunolabeling prior to fixation (Kireev et al., Nat Meth. 2008, 5:311). It's a bit tricky and works only with monolayer cell culture, but the results are avesome!
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