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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalGreetings!
I am fixing monolayers of cells on coverslips that have the "appearance" of
being sheets of cells perhaps overgrowing one another. Confocal study did
not show more than one layer of cells.
I fixed the cells in 3.7% formaldehyde followed by methanol/acetone
perforation. I used aquapolymount for mounting.
Perhaps you all could help me with some questions:
Would the fixation method cause the cell layer(s) to collapse? What is an
alternative?
Is it possible for the cells to appear crosshatched and not be overlapping
significantly - just cytoplasm perhaps? I was trying to tell mainly by looking at
nuclei.
I've seen a lot of talk about building inserts to prevent the cells from being
compressed - and have also been told it doesn't matter. What's the opinion
here?
If I use nail polish on freshly mounted coverslips, or even after they've "set"
for an hour or two, unpredictably the end result is that the cells are sucked
off the coverslip into the polish. Is this the brand of polish (hard as nails) or
some result of a combination of polish and mount solution? Has anyone else
had this happen? Is it an excess of mounting solution?
I would appreciate any help here - I'm pretty new to imaging.
Thank you,
Valarie McGarty B.S.
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