Hello everyone.
My question regards the mounting stage in the immunocytochemistry protocol.
I use 10ul drops of Flouromount Aqueous Mounting (Sigma, kept at RT) to mount 13mm coverslips with fixed neural primary culture (I always make sure I dont have any babbles).
After mounting I let the slides dry over night and in the morning I store them at 4C for few days until I take the pictures.
It seems that some of the mounting solution drys so much that it tears the cells, and in other cases it seems as if I did have plenty of babbles when I mounted, even though I didnt.
Any suggestions?
Thank you!
Maya
There is actually no way to prevent aqueous mounting media from drying out and cracking. Try slowing down the drying process by keeping slides in a humidified chamber. For this reason we are using a glycerol-based mounting media from Neuromics (https://www.neuromics.com/SF40000-10). After mounting slides need to be stored in a freezer and last for up to 1 year. The drawback is that it will never dry out so slides have to be stored in horizontal but not in vertical position to prevent leaking of the media.
Hope this helps.
Hello,
in our lab we usually do lot of IHC and ICC, both cases we use dako fluroscence-mounting-medium and it just work really good. why don't use little more amount mounting media and try to dry in a humidifier chamber or in a place which is not too warm. i attached the link of product , if you want then can have a look.
https://www.agilent.com/en-kr/products/immunohistochemistry/ancillaries-for-ihc/mounting-media/fluorescence-mounting-medium
https://vectorlabs.com/uk/vectashield-hardset-mounting-medium.html
Vecta do a range of hard setting mountants for IHC-F with and without counterstains such as DAPI & Phalloidin.. However, you could always seal (ring) the edge of your coverslip. Fast drying nail varnish is good (I use either a "Goth Black" or a "Barbie Pink") or you can use DPX or Pertex but they don't dry as quickly as the nail varnish... You may want to use a larger diameter coverslip if you are "ringing" the edge.
always I use humidifier chamber for IHC protocol in addition to seal the edges of cover slip with nail varnish
I would suggest you to seal the corners of your glass coverslip with colourless nail polish so that mounting medium doesn't leak out. Also, you can keep these sealed coverslips in a dark box at -20 or 4 degree celsius for long term storage.
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