I would like to know how to mount the coverslips on glass slides? I culture 293T cells on coverslips for ICC. I want to fix them with 4% formaldehyde. After fixation, should I use any fluid as a spacer between the coverslip and the glass slides?
Do you use gelatin-coated slides? Do you use fluorescence? Maybe mounting medium:
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Thanks Christian Bravo-Rivera for your answer!
I use Mitotracker DeepRed to stain mitochondria. No, I do not use coated slides. I culture the cells in 100mm dish containing coverslips. After they reach the proper confluency, I add the Mitotracker (diluted in PBS) and incubate with for 20 mins. Then I aspirate off the Mitotracker and add 4 % formaldehyde (15 min RT).
After these steps I would like to mount the coverslips on glass slides, but I am not sure wheter I should use any fluid between the two layer.
In nutshell:
Coverslip - fixed 293T cells - ??any fluid?? - glass slides
You will definitely need mounting medium, to keep your samples hydrated and stuck to the slide. Since mitotrackeker deep red is a fluorescent dye, a mounting medium with anti-fading properties could be useful if you want to keep your samples for long term. We use the Dako Faramount Aqueous Mounting Medium, I can only recommend it. If you analyse your samples straight away and don't need to store them, then preparing your own medium can be an option too, for example by adding gylcerol: PBS in a 9:1 ratio, adjusting the pH to 8.5-9 (apparently this helps to retain fluorescence). This will not solidify so you will want to keep your coverslips on the slide by putting some nail varnish around the edge. This is just one solution, but there are many others if you need to make your own. Sok sikert! :)
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