My colleague grew some cells on coverslips for me to do FISH and IF staining. This is my first time staining this way. Can anyone share their method with me? What I mean specifically are the mechanics of the staining, not the protocols, i.e.: do you lay the coverslip flat on a kimwipe and pipette each solution on? Do you keep it in the plate it was grown in? I feel like the solutions are getting underneath the coverslip (in-between the coverslip and the bottom of the well) and it's a bugger trying to get the coverslip up to tilt it and rinse it off. Sorry for the rookie questions, but I've never seen this done and can't seem to find any tips online. Every link seems to take me to actual protocols. Thanks in advance!
Try putting them over a piece of parafilm. It should do the trick. Best of luck,
D
Dear Adrienne,
You can use chamber slides for this. This is an easy method for staining protocol after cell culture. I added two pictures about chamber slide.
Good luck
Dear Adrienne,
I keep the cover slips in the chamber the cells were grown in (i. e. in well-plates). The disadvantage is that you have to shake the plate (use an orbital shaker or similar device) in every washig step so that the solutions under the cover slip will be removed. By gently pipetting the solutions at the edge of the cover slip, you also might rinse the underside.
To remove the coverslip once you are finished I use a fine forcep (Dumont No. 5), but you should be careful not to flip the coverslip when doing so.
There is a big disadvantage doing ISH/FISH with a cover slip placed on parafilm: The parafilm will melt during hybridization due to high temperatures. Don´t use Parafilm! (I´ve once tried to seal a moist chamber with it for ISH, it was a mess!). Normal cell culture plastic ware will be stable at temperatures around 60°C, so keep the slips in the wells (use a moist chamber to prevent drying!).
For IC, parafilm should be ok.
Thanks everyone! I did use parafilm (for all steps except for the hybridization and post-hyb solutions since they are at high temps) and it was really helpful. I'm finding it extremely difficult to get the coverslip out of the well, even with a fine forcep so I will look into the chamber slides. Thank you!
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