Hi Laura, Many years ago I have cut vibratome sections from unfixed tissue in agarose gel. I needed 50 um sections and modified the method of embedding to shorten

embedding time(because of tissue being unfixed) by using one of the old glass chambers (for embedding block material) connected to a pump). I do not have a protocol, but thought that you might try experimenting on some trial tissue. I have checked the thickness of sections on a microscope with Z-axis movement and it was consistently 50 um with minimal variations.

I have tried to find some publications as I remember reading at the time some papers on unfixed tissue/vibratome sectioning but couldn't find full papers.

The reference below may, or may not be relevant - agarose gel embedded tissue (fixed/unfixed cut at 5-=100 um)) - because I can't get full paper as my university doesn't subscribe to the journal.

https://www.tandfonline.com/doi/pdf/10.3109/10520299309105642?needAccess=true. If you get your project working - could you possibly let me know please? I do not work in this field now - just FFPE biopsies with immunohistochemistry - but I am still interested in methods dealing with tissue processing. Good luck with your project.

Maria Jeziorska Thank you very much for your help. Niether I could find lot of paper on it. However, I didn't read the one you sent me, so I will do it carefully.

If I success I will of course let you know.

Thank you again!

Dear Laura,

embedding in agarose would be definitively a way but it is still quite difficult to cut 50 Micron sections from unfixed tissue. It takes a lot of work.

Good luck and best regards, Daniela

Hi, I have memories although the technique I used is somewhat different. Using fresh tissue we applied cooling or freezing. Fast freezing with N2 and then applying OCT (fluid surrounding and infiltrating a bit) and cut it in cryostate. Thus you can have 10 u sections or less, but probably vibratome should do it too. The temperature (-20--26 C) is warmer for thicker sections. In the US whole brain sections worked nicley. However all these may be useless if you have to remain on room tmpr. Yours Laszlo

Laszlo Havas thank you for your help, however I have to keep the cells alive, that's why I want to cut with the vibratome and not the cryostat.

Daniela Rodler I gave it a try yesterday without embedding in agarose and the results were quite encouraging.

Thank you all ! =)

Maria Jeziorska as promised, here's the way I proceed to obtain thin slices with the vibratome.

First of all, I think it is really important to keep the cutting medium you will use in the ice in order to cool it. I also surround the cutting vat with ice.

Finally, in order to promote maintenance of the extracells junctions, I add Mg2+ and Ca2+ in the PBS (my cutting medium)

Hope it will help.

Again, thank you all for your advices.

Hi Laura, thanks for the update. I was cutting on vibratome smothered in ice, the medium also cooled, so the same conditions. The vibratome method is different from cryostat method. I have used both and at different conditions and temperatures. Vibratome allows for avoiding micro-fractures and supporting agarose gel nullifies ‘squashing‘ effect - so these are pros for vibratome.

if you do not snap freeze your tissue but choose a slower method with isopentane as interim step and adjust temperature in cryostat for cutting to the best for your particular type of tissue - it makes a cryostat method a good choice too.