Hi,
I am currently planning on setting up a pre-embedding immunogold TEM experiment in cell culture.
I've had no experience with EM so far, and so I was hoping to clarify some questions regarding the protocol (this one assumes cells are seeded and stained on coverslips, and that these are removed only after embedding):
1. The type of coverslips used for this protocol was not mentioned. Do I need to use glass or plastic coverslips? Are some special coverslips needed for this method?
2. Will poly L-lysine coating of coverslips interfere with subsequent removal of cells from coverslips?
3. According to the protocol, samples should be post-fixated in 2% glutaraldehyde - how long can I keep the samples in the fixative?
Thanks
I suppose you will not prepare (embed, cut, etc.) your specimens by yourself. So, talk to a person who will do it. There are no a single best protocol. Some people prefer glass cover slips as a substrate, some (like me) prefer plastic (like Thermanox). So, leave decision to your TEM person.
Thank you Yvonne, that sure helped.
Vladimir - thank you. I have tried checking with my TEM person, but so far I've got the impression she is not that familiar with the staining part of immunogold protocols... will try asking her anyway.
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