Immunostaining- opaque layer on fixed cells
Posted 14 July 2009 - 08:14 PM
I've been having lots of trouble fixing and imaging my immortalized neuronal cell lines. Every time I image it with confocal microscopy or fluorescence microscopy (FITC and Texas Red) an opaque layer of liquid covers areas of my coverslip. There are some areas that do not get covered and the cells are fine (distinct neuronal morphology)...but for the majority of the coverglass the cells are covered in this "layer". I use VECTASHIELD with DAPI mounting media. If anyone could please shed some light onto my problem I would be very very appreciative.
My protocol is basically allowing my cells to uptake FITC conjugated to a molecule and then fixing it. After fixing I stain again for another protein in hopes to colocalize. I use 4% PFA to fix my cells for 20 min at 37degrees celsius and wash many times with PBS.
Posted 15 July 2009 - 03:00 PM
If you hold up your slide, do you see the opaque area? ... Are you sure that it's not just dust or something? Take a lint-free wipe and wipe it down, perhaps with a touch of methanol to make sure it's not something fixed to it. For a slide to become opaque is kind of strange. Do all your slides do this? Just some? Is this just one or two isolated incidents?
And yes, a picture would be great.
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Posted 15 July 2009 - 05:38 PM
What do you think?
Posted 16 July 2009 - 04:22 AM