Hallo Alexandra,

what do you exactly mean by rosettes? Do you eventually have a picture of that?

I am working with OHSCs from p2-4 mice, but we do not embed the slices in agarose. We slice them with a tissue chopper at 375nm and then culture them for weeks on membrane inserts and they do not form any strange things. Maybe the agarose is the point.

Greetings!

Hi Natascha,

Thanks for getting back to me!

I've attached a file to the question showing the 'rosettes', I think that is the best way to describe them. You haven't seen anything like them? I also thought the agarose could be the source of the problem. A colleague mentioned using a tissue chopper, but I wasn't sure if perhaps my tissue would be too delicate for it. 

Also, do you see any differences in expression patterns when you culture your slices? In a few of my slices, I have seen expansion of the expression patterns of some of the marker genes I am using, but cannot see a reason as to why it happens in some slices but not in others.

Kind regards,

Alex

Hallo again;),

I read your question again, and I noticed that you are using the whole brain, I thought that you were doing also hippocampal slices. We are only dissecting the hippocampus and slicing it afterwards, this is no problem with the tissue chopper.

Why are you using embryonic mice? I can imagine that it is very difficult to slice these tiny brains with the tissue chopper as the tissue is very soft, I would say.

As we are working with hippocampal slices, I usually do not have cortex around and so I do not see any rosettes like you in your picture.

We are focussing on neurons and glial cells and during the culture time we see, that the slices themself do flat over time, but when we immunostain them we do not see so much differences in cell numbers.

I am sorry, that I cannot really help you :(.

Good luck!

Hi,

Ok, thank you! The slice in the picture was not very healthy at all, but it showed the rosettes. I'm using embryonic because I'm looking at the onset of neurogenesis in the cortex, we've found the cultures much easier to do at later embryonic ages, but for this experiment I need use young (E11.5) embryos.

Thank you for your help, I've spoken to a colleague again about the tissue chopper and she will let me watch her using it next week, although it might not be suitable for our tissue.

Kind regards,

Alex

The media can make a massive difference to the health of your tissue. Maybe try doing a screen with different types of media (do a literature search to see what's been used before - try Neurobasal Med if you've not already). I had to do the same for spinal cord slices, and I found this resource really helpful: http://dx.doi.org/10.13070/mm.en.3.175

Hi,

That's a good point, I'll have a look into it. Thank you for the resource, it's definitely interesting reading,

Kind regards,

Alex