I am FAC sorting testicular cells and I would like to do FISH to tell the spermatogenic stage. Some populations are very rare ~1,000 cells, so I wanted to sort them straight onto slides and do the fixation and staining there (sorting in tubes and cytospin would mean sample loss and the cells would be very dispersed on the slide after that). I have gone through so many protocols that don't really work for the above conditions.. Any suggestion?
Hello,
Generally you can perform FISH on slides as you would with filters (many protocols out there, like those from Pernthaler). Important is, that you fixate your cells on your slides! That is usually easier with Teflon coated (or similar) glass slides, those also come with little indentations. Another important part, is that you get the conditions for your probes right, like temperature and formamide concentration. Are you using a fixative before sorting?
Best regards!
Hi Jennifer,
Thank you very much for your reply. I am not using any fixative before sorting (at least I haven't used any so far). I was thinking of sorting on adhesive pre-coated slides and my main concern is the fixation afterwards and before the FISH.
As for the FISH protocol, I do have one that has worked for us in the past, but we never did it for cells but only for tissues.
Hello Chrysanthi,
Maybe you have to try which approach might work better, a fixation with paraformaldehyde before sorting or once your cells are on the slides. You can also do the fixation on the slides, just ensure your cells adhere to it. In principle the protocol for tissues and for cells (on filter) should be the same. Maybe you can have a look here https://www.researchgate.net/publication/246206760_Fluorescence_in_situ_Hybridization_FISH_with_rRNA-targeted_Oligonucleotide_Probes. Still find this work by Pernthaler et al. very helpful.
Best regards!
Article Fluorescence in situ Hybridization (FISH) with rRNA-targeted...
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