You could try cutting the sections a little bit warmer than usual. Warm up the tissue in the cryotome first from -80oC to -20oC and then I usually cut cryo sections at -20oC but with fat (breast and brain) I cut a little warmer around -15oC. With brain I cut thicker sections around 8-10um and I find this helps too. Another tip is to surround the tissue with OCT and this will help keep the tissue together when you cut.

Hope this helps

-80C

but at once after -80C sections are bad and are presented by multiple folds. So, we cool samples to -20C, and after that we perform cryotoming.

Did you try sucrose fixation of the samples? At least in brain tissue it prevents the "squooshing" of the tissue when you cut it. I usually fix the brain in formalin (4% PFA) and then in sucrose (30% till the tissue sinks) before freezing the tissue. Then you can make frozen sections. I hope this is helpful. Good Luck

That is correct because fat can only being cut in a cryotome at -40 degrees. The disadvantage is then that the other tissue will scatter/splinter. So or you cut away the fat when this is possible or you remove the fat via an pre-impregnationstep of aceton and then freeze the tissue .... otherwise it will become a compromise in cutting-quality between the fat and the other tissue. Good luck Henry

other option is dry ice with acetone, the freezing is immediately. Good luck

You could try cutting the sections a little bit warmer than usual. Warm up the tissue in the cryotome first from -80oC to -20oC and then I usually cut cryo sections at -20oC but with fat (breast and brain) I cut a little warmer around -15oC. With brain I cut thicker sections around 8-10um and I find this helps too. Another tip is to surround the tissue with OCT and this will help keep the tissue together when you cut.

Hope this helps

Samples can be embeded in OCT and then freez in liquid nitrogen immediately after sampling. You can store your samples in -86 oC and cut cryo sections at -20oC.

Collect each section on a strip of adhesive tape, then freeze dry it.

Dear Evgeny V. Denisov,

Interesting thread but unfortunately for best help we don't know some important parameters like:

i) samples prefixed with buffered FA or another fixative or native tissue?

ii) freezing samples (which dimensions?) for (only) routine light histological / histochemical examination (routine H&E and eventually LH-IHC-stainings)?

iii) frozen tissue used for further, extended research research (mol.biol., mol. genetics, electron microscopy)?

iv) not knowing whether you have frozen the tissue samples initially with LN2 (liquid nitrogen) or just placed into - 80 degr.freezer (physical matter: recrystallization point around minus 85 to minus 80 degrees C.).

v) the question arises whether you would be able to follow Henry Dijkman's proposal to eliminate fat tissue as much as possible prior to freezing (thus perhaps eliminating also micronodules... or other structures important for diagnosis).

OCT "embedment" always favored prior to freezing (also for orientation / oriented sectioning of sample after frozen storage).

Always wrap aluminium foil around your frozen specimens when stored in the freezer.

Interesting and valuable tip from Maree Gould, but perhaps erroneously got a "downvote"! from somebody who could easily reverse this action if it was not intended definitely!) .

Naturally - from a physical aspect - it would be of favour also to soak tissue in gradually increasing Sucrose-solutions after fixation and prior to freezing (penetration! will depend also on dimensions of specimens!).

Short question to Henry Dijkman: concerning your statement: : is this your experience or is there any serious reference for this we should know about?

And as the last question I would like to ask honestly all of my pre-posters: any difference (experiences) in sectioning outcome using a variable/different knife temperature (say minus 40 degr. vs. 150 degr. C KNIFE temp, minus 15-40 degr. C sample temperature?).

Should be glad receiving at least some answers to my questions....

Best wishes and good luck!

Another note added (concerning freezing, OCT-compound, sucrose gradient as cryoprotectant): see ResearchGate: https://www.researchgate.net/post/Cryoprotection_with_sucrose_30_after_neutral-buffered_formalin_NBF_fixation?cp=re65_x_p2&ch=reg&loginT=j9gkHiwNf2w2gjWkihARRzrO87jCIepI&pli=1

best regards, WM

Thank you very much for your attention and your suggestions!

Some refinements:

1) samples are not fixed, after surgery they were placed in liquid nitrogen and then in -80C

2) samples will be stained by H&E and used for laser capture microdissection

3) the microdissected material will be used for DNA and RNA isolation

4) fixation in formalin is not possible because of negative influence on DNA and RNA quality.

And questions:

1) How acetone influences on DNA and RNA? Can I fix already frozen samples in acetone? What protocol should I use?

2) And such questions about OCT: effect on DNA/RNA, possibility of embedment of already frozen samples, and protocol.

OCT embedding is ok for RNA. We've done a lot of those kind of work. Since RNase is everywhere, you have to be careful to control the activity of it. RNase is not so active at -20~30C. So you don't need worry about when you embed or cut. But you will need fix the slides ASAP after you cut the slides with either cold Acetone or 70% Ethanol to preserve the RNA. U'd better perform the LCM ASAP to get better quality of the RNA. Otherwise, the slide must be store at -80C. Qiagen and Leica both have very good protocols for LCM sample preparison .

Yes, I use Qiagen kits for RNA isolation. It allows to get RNA of good quality. However, recently, I have a problem of cutting of frozen breast tumor samples, which are penetrated by fat tissue (as mentioned above). Taking into account your suggestions, I will try to use OCT embedment or acetone fixation of samples and already then to prepare sections by cryotome.

Dear colleagues,

I have one more question regarding the protocol of aceton fixation. How long should I fix samples in aceton, overnight or some minutes? What should I do after fixation? Can I cut samples immediately after fixation? Or I need to perform further procedures, e.g. any washing and embedment (paraffin)? In general, I would be grateful if anybody could share the protocol.

Thank you in advance!

You should never fix the sample with acetone before you get the sections. Acetone fixation should only happened after you cut you sample. The order should be: Embed tissue in OCT, Cut frozen section at -35-40C, Fix the slides in cold acetone/ Ethanol, Stain the slides with H&E/ Crystal Violate, Perform LCM, DNA and/or RNA extraction, Downstream analysis.

Thank you, Zhijiu!

Dear Denison, section of fat fresh samples is not easy to cut. This should be done in very low temperature.

One possibility to cut those samples is to prefix them in 10% formalin + ClCd 1% , wash in sucrose, frozen and then cut in cryostat . The adoption of this procedure, however, depends on the approach that will be adopted in the study.

Thanks Mahdy. Old methods sometimes still Works !

Thanks, Telma! I'll cut in very low temperature. Formalin is bad for RNA.

Use very low temperatures as Telma suggested. Also cut thicker sections. For LCM I go with 10µm as a minimum. For fatty tissue I have had to cut as thick as 20-25µm to get reasonably intact sections. What is most important is what kind of cells you want to microdissect. If it is tumor or blood vessels, then you should be able to see them even if the section looks awful. I usually cut a test section, stain quickly with hematoxylin and immediately look under a microscope to determine if the structures I am interested in are present or not. This takes less than 5 minutes. Another important thing to remember is to use a very sharp blade. Fat sticks to the microtome blade so it has to be cleaned carefully or a new section of the blade has to be used each time. Please let me know if you need any parts to be clarified further.

Thanks, Sabyasachi, for your help! I tried to cut at low temperature (-35C) and I did not feel the difference. Of course, fat samples were cut, but sections (5 um) had many folds and were unsuitable for LCM. Tomorrow, I will try to cut thick sections and to use sharp blades.

Hi Evgeny, I would suggest that you do not use 5µm sections for LCM. Go with 10µm as a minimum. Also, if possible, orient the tissue in such a way so that the fat does not come in contact with the blade first. Could you let me know what cells/structures you want to microdissect?

Thanks, Sabyasachi! We will try to cut 10 um sections. As for cells, we isolate different architectural arrangements of tumor cells from breast tumors.

Evgeny, did you examine the sections under a microscope? Even if it looks bad the tumor architecture is usually preserved.

Dear Sabyasachi, sorry for late reply! I tried 10 um sections, but it is not our variant. Tumor architecture was so bad that morphological structures, which we isolate by LCM, were fuzzy.

Sorry to hear that Evgeny. Did you talk to your Institute's histotechs regarding this?

Cutting and staining sections are performed by our histotechnicians. And they agree that 5 um sections are good for my project; however, they do not have any ideas regarding cutting fat samples (((

Dear Abhijeet, thank you!