I would like to scan calcium-indicator-dye-loaded neurons after fixation, just to know, which cells have been loaded. Fura and Carbodiimide fixation have been reported to retain fluorescence but I prefer paraformaldehyde fixation for several reasons. Is there a single calcium dye, which is not quenched after paraformaldehyde fixation?
Sabine, I think there is little interest in measuring free Ca levels in a fixed tissue, as it depends solely on the [Ca] of the solution you have your sample in (even mild fixation breaks down the membranes enough to equalize concentrations very fast). I realize this is not an answer to your question, it's just a warning that even if you got your answer you still wouldn't be able to measure the intracellular [Ca] in fixed cells/tissues because of membrane permeabilization.
Peter, Thank you a lot for your answer. I am quite aware of that membranes brake down after fixation and we do not attempt to measure free calcium in a dead celll. We simply would like to see the cells, in high resulution after fixation, which have been observesd in calcium imaging, before fixation, while still alive in an whole, intact and still living insect brain. Any suggestions?
No problem, sorry for being so slow....
When we did something like this (measure [Ca], then do immuno on the same cells), we used a short, low-temperature methanol/acetone fixation after Ca-imaging in the cells. Then we would "just" go and find the same spot. This is not a very elegant way to go, you get only few results with a lot of work.
I've never worked with insect cells or tissues so I'm not sure how easy or difficult it may be to express new proteins in them - but using Ca-indicator fluorescent proteins may be one possible way to go. I know from experience that GFP-related proteins are fixable with 4 % PFA and retain their fluorescence. Look into the GCaMP literature. The nice thing with these is that these contain an eGFP subunit which probably retains it's fluorescence after fixation, and if the signal is too week, you may be able to detect them with IHC (with an anti-GFP antibody). All you need to do is to get a suitable vector (probably a virus more or less specific for infecting the cell type you are interested in) with GCaMP 5 and injecting it in the living insect. Wait for GCaMP to get expressed and there you go.
In ours hand Calcim green-1 dextran (Molec Probes) works!
All the best
Alejandro
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