My lab has been attempting to work out a fluorescent in-situ protocol for fixed, frozen, rat brain sections. We're almost there, but the main issue is that whether the tissue remains adherent to the slides seems like it's basically luck. One week I'll get beautiful results, the next week half the tissue sections lose our region of interest. Hoping for tips to keep it more consistent.
- Animals are perfused with 4% PFA, brains are flash frozen, sectioned at 20 um on microtome, kept in antifreeze.
- Before mounting on slides sections are washed in KPBS 6 x 5 min each, then mounted on Fisher Superfrost Plus slides in KPBS.
- Slides are dried and then placed in a vacuum chamber overnight (~18 hours). We have also tried 48 hours, still had some issues.
- Postfix in 4% PFA for an hour, then 5 x 5 min KPBS washes. This is usually when tissue starts to lift up a little and fold at some parts.
- Protease treatment with various solutions, tissue tends to fold up a little more during this as well. After the entire in-situ protocol, entire chunks of tissue sometimes tear off (often times thinner areas of gray matter). I think it is a mechanical issue from poor adherence and repeated washes in different buffers, not any chemical problems.
Any suggestions for improving tissue adherence welcome. Thanks!
Hi Alyssa,
fixiation with PFA could lead to a shrinking of the tissue. Since you're using quite thick sections this might be the reason why the tissue detach.
If possible, depending on your experimental onset, you could try thinner (10um) sections and in addition to PFA use Glutaraldehyde (Karnovsky's Fixative).
I would also decrease the time of post-fixiation to 15min instead of 1h.
Best
Lukas
Hi Alyssa.
We perform double FISH in our lab with the same protocol you described. But with two differences: i) we use 14 um slices (in the past we used 20 um slices and we did not have this problem) and ii) our post-fixation time is 20 minutes in PFA @ RT (since animals are perfused I think maybe you do not need so much time in post-fixation).
PS: sometimes I found that some batch of Fisher Superfrost Plus slides are not as much adherent as they should.
Hope this works for you. Do not doubt to contact me if you have any extra question about FISH.
Thank you all for the suggestions. We will probably try the thinner sections and slide charge enhancer first.
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