How long tissue can stary in sucrose or fixative before cryosectioning? - (Jul/30/2008 )
I have to store my tissue a couple of weeks before they are cryosectioned. I'm wondering in which solution they can stay happier? fixative ( 4% paraformaldehyde in fridge) or sucrose 30% at room temperature?
Prolong exposure to fixative more than 3 days can make tissue brittle and hard to cut. I would suggest you stop at Sucrose stage if you must do so.
Definetly stop when your tissue is in sucrose. And I've always learned to put it at 4 degrees if you save it for a longer period of time.
I have a related question... We've fixed lung tissue (in 4 % PFA) and put it in 30 % sucrose o/n. But they won't cut on the cryostat!
We think that it's because the lung tissue isn't perfused w/ the sucrose yet, so my question is... any way to speed that up?
Ow...Just so you know, all my other tissues that I've done the same process (48 hrs in 4 % PFA and O/N in sucrose) are fine for cutting.
We can actually see the tissue is pushed by the knife iso being cut. And when we put the temp down (to -40) it cut thick and thin, so no nice section. That's why we figured it would be the PFA in there.
I would love to hear about any tips or tricks in this case.
Unfortunately I don't have any experience in tissue sectioning since it's done by the technician in the lab ... but have you tried other fixatives for this specific tissue?
Ddkb: I dont know if the lungs were inflated with fixatives as well. If there is air space left you will have difficulty cutting the lung spec.
We haven't tried other fixatives. Mostly because we're going to work with bugs and we already have a protocol for 4% PFA fixation. But any suggestions, I'm definetly willing to try out
The lungs were perfused with the 4% PFA. It's what I did for the paraffin sections as well and of course that worked fine! Not with the cryostat though and that's the technique I'm interested in. So there shouldn't be any air space left, but I figure it might have been harder for the sucrose to get into all the lung tissue when it's perfused, so there's probably still some PFA in there, which I thought might cause the trouble... See I'm not that experienced, but that was my 'logical' way of thinking.. I concur, it's not always the 'right' way of thinking about something.. haha!! But you've got to start somewhere!