Hi all,
I'm facing problems in staining the cryosection of the cells encapsulated in hydrogels.
I'm using both charged slides as well as the albumin coated slides for the HnE staining. During the washing step, the sections are floating and washed away.
Can anyone suggest me the protocol for hne on cryosections?
Thanks
Nisha
Try this:
Take slides out from freezer and lay flat on Room temperature horizontally. Leave the slides for 30 minutes so that sections sinks at the bottom after melting the OCT. That will help sections to attach to slides better. Sometime if we start immediately, there is OCT left between section and slide, that will cause sections to wash off.
Best,
I tried to keep them for 5 min at RT after taking the slides from the freezer. Once it comes to RT, washing step makes my samples go away.
Try longer time (if you are fearful of drying section, use buffer drops on sections and also try to suck the melted OCT with kim wipe if you can see the sections) so to clear OCT layer between section and slide; and section gets to slide surface to attach. That is critical, I used to lose sections due to hurry. Hope it works.
Is it recommended to fix the samples before freezing or after making the sections to fix them?
I do not know the difference. But I always fix tissue in 4%PFA for 1hr at ice or 4C, than wash with PBS 2 times and pass thru 15% sucrose and 30% sucrose till tissue sink (it takes 24hrs depend on thickness of tissue). After sections are done, I use few drops of 0.2% glutaraldehyde (cold) for 10 minutes on sections to fix it again. And than proceed--. They say sucrose treatment preserve the cell structure better.
Everyone has slightly different method of fixation. Some people throw the tissue in OCT in the block and than throw in LN2 and they probably fix sections later, they do not use sucrose treatment, it means.
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