Hi,
I have a problem with cryosections while staining. Positively charged slides are used for cryosections, in spite of that during the staining process, the sections are washed away during the washing steps. Please help me in sorting the issue.
Thanks
Nisha
Hey Nisha,
I've had the same problem and it turned out to be a combination of factors.
1. Charged slides only work if your sample is of the opposite charge or close to neutral, make sure the pH of your buffers is correct before you mount your sample, including your fixative if you've fixed before cryosectioning. Also very important is removing any fixative from your sample before sectioning, so don't skimp on washes.
2. Make sure you allow enough time for your sample to dry onto the slide, for me that turned out to be at least 1 hr on a hot plate set to 37C.
3. Sample thickness can be a problem, if too thick it can detach, I recommend staying at or below 25um.
4. If you're doing immunostaining, consider whether you can free float your samples instead, and just mount at the end.
If nothing else works, consider making your own subbed slides, that was how I managed to save my samples that had improper pH.
Good luck!
Hi Nisha,
I have many years of experience with frozen sectioning and IHC. Would you please include your fixation, embedding and staining protocol.
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