Hi Guys,
I’m having a terrible time trying to perform immunogold pre-embedding transmission electron microscopy, on monolayer cells to do CLEM.
Basically, I follow the protocol suggested in many papers by Mironov, Polishchuk and co-authors (nanogold labelling followed by gold enhancement).
My problems are concerning mainly a widespread background (small spots on all the membranes) in negative controls also!!!
I used many different primary antibodies, but the results are more or less the same.
I would really appreciate if someone could suggest me a good protocol.
Many thanks!
Francesco
Buongiorno Francesco,
You find a speckled background signal after the immunoEM staining with nanogold, followed by gold amplification. You might want to share one or more (details of) images where this problem occurs.
A: Do you find a good signal at places where you expect the signal? In other words, does the staining work and is the nanogold conjugated 2nd Ab work fine?
B: How do you perform the gold amplification? Self-made from AuCl4 or commercial kit?
Hard to tell, there is so many things that can go wrong! From my experience the primary antibody is the single most critical thing in IEM. Make sure you have tested and optimized this antibody in immunofluorescence experiments thoroughly before going to the EM level. Some antibodies are simply not suited for IEM labeling.
If the primary is fine: Do you observe the background labeling after silver enhancement only in combination with the nanogold (with or without primary), or even when the secondary nanogold is omitted?
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