Hi
I usually perform immunofluorescence on cells grown on coverslips. But this time im growing them on transwell inserts. All the fixing and antibody staining/wash steps will be soaking the transwell into wells containing the respective solutions. However i am not sure if my mounting protocol would fit the transwell membrane.
What i intend to do is after fixing/permeabilization/antibody stainiing/washing steps, i will cut the membrane of the transwell insert then place it on a glass slide with cells facing upwards. Now this is the tricky part, the mounting media i normally use is not the 'gooey sticky' type but rather almost like water (if you can imagine- Mounting medium is in attached picture) . If i add drops of this watery mounting medium onto the cut membrane and then place coverslip over it, will it 'stick'? It has to stick. Also, im not sure if the transwell membrane thickness will be too much such that the coverslip and glass slide cant even touch one another.
If you are not familiar, i will still appreciate any comments/suggestions.
Hi Iwan
If I infer correctly, you don't normally seal your coverslips when you mount them on the slide? We paint around the edges of coverslips with nail varnish or VALAP after we mount them. Not only does it keep the coverslip in place, it also limits oxygen diffusing in, helping to prevent oxidation of the mounting medium. That way you don't have to worry about the mounting medium being sticky or not.
For in vitro cells on cover slips, after staining we mount them facing the microscope slide. We use Aqua-Poly/Mount from Polysciences as a mounting medium for both the in vitro cells as well as for tissue sections. The medium is water-based, not messy, not toxic. But a bit pricey.
Although not tried IF on transwell myself, I will guess the membrane like plastic coverslips will be autofluorescent, so I would not use this technique here, but enzyme based (hrp or ap). Then you can dry/dehydrate and mount permanent with xylene based mounting media (Entellan, Histokit etc).
Hello
Link and discussion here will help you
Good Luck
https://www.researchgate.net/post/According_to_your_experiences_what_is_the_best_cover_slip_mounting_medium_for_IF
http://sydney.edu.au/medicine/bosch/facilities/advanced-microscopy/user-support/Mountants.pdf
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