There are multiple things you may want to double check. 1. the thickness. 2. the speed you let your blade passing your tissue, sometimes you need to let the blade cut into the block with a steady speed, then cut through the tissue with a relatively fast speed. 3. the blade condition, every cut, you need to carefully clean up your blade, use the side of brush with a rolling motion to gently clean up the blade, the rolling direction is not against your blade. if your blade dent, you will definitely lose your material. 4. Same as Mohamed's reply, keep everything in the chamber. Things here I mentioned still need your optimization for yourself. Hope this would help.

I would make sure the temperature you are using is right. Sometimes a variation of a few degrees can alter the quality of your sectioning. Another factor is the kind of samples you are presently using.

Try changin blades, make sure all materials you will be using are at the right temperature, and you should be able to sucessfully get good sections.

It often helps to dry the tissue by blotting gently before embedding in OCT, I have found this helps to prevent tissue falling out of the OCT block.

If you warm the cutting surface of OCT for a second by hand, you may be able to get sections, when you use a sharp knife and appropriate tempatatre of the knife (-20--25C),

One important fact could be the kind of slides you use. I recommand to use super frost plus. And now you can try different temperatures.a) put the slides on a heating plate at 40°c, b) put the slides in the Kryostate chambre or c) use them at room temperature. If all this wouldn't help., don't embed the tissue in OCT, just fix it with a drop of 30% succrose on the tissue holder and cut very slowly an carfully.

Good luck!

Ute Neubacher give the above answer ,i share with him what he advise as solution to the problem of Rosa.

There are a number of factors you need to optimise. Firstly, the optimium cutting temperature will be dependent on what tissue are you sectioning; solid, homogemeous tissues, for example liver, are best cut at relatively warm temperatures (-13 to 15C) whereas tissues containing fat, e.g. breast, need to be cooled down to appprox -25C). Trial and error will find the sweet spot for your tissue, although you do need to leave the tissue for 30 minutes to equilibrate after changing the temperature.

As previously stated, the quality of the blade is important, but so is the cutting angle. I usually find an angle of 4 or 5 degrees to be best.

You have to make sure the anti-roll plate is adjusted correctly so that the tissue just missses it on the upstroke. Make sure it's free of nicks & debris too.

Finally, it might be worth trying to cut your tissue "naked", i.e. without embedding it in OCT. Try fixing a piece of your tissue directly to the cryostat chuck with a small blob of OCT and cutting the tissue alone. I cut almost all of my cryosections this way and very rarely embed my samples in OCT.

I am not sure if your sections are floating away from your glass slide (in this case you could use glass slides coated with poly L-lysine) or that the tissue block is lossing its volume after embeding in OCT (as Brian suggested you should gently blot it and also make sure that the sample is kept for several minutes at about -20 C before embedding in OCT). Good luck!

I washed previous fixed samples in "tissue wash" a phosphate buffer (no sucrose), then mount on a pedestal of frozen OCT and cover with OCT. I still think the type of slide is probably your best bet as Ute suggested. I use the same type and have no difficulty with this system.

Check if the anti-roll is properly adjusted. Depending of the type of tissue you can froze it quickly by immersion with cooled isopentane (-80°C) and then glue it to the holder without need to enrobe in OCT. Then observe carefully the slice of section when you cut it (if it sticks to the rear of the antiroll or close to the blade) when adjusting the antiroll. Check also the blade and the angle of cutting. Take care of completely immobilise the holder with your sample.

"glue" it with isopentane of course!!

Dear Rosa, You have not mentioned, how did you wash your slides to remove OCT? Are you using the anti-roll? May be you need to think our method of slide washing, which may loose the sample. Yes, I agree with one of the answer above, you need to use super frost or Poly lysine coated slides.

For animal tissues use at least 5 mm3 specimens. Freeze in n-hexane precooled to -80 for a min, immediately on the speciment holder. No embedding medium required. Adjust speciments for 30min in cryostat temperature (-26 degrees). Cut 5 μm thick sections. Use any clean slide at room temperature to take the sections. Leave the slides out of the cryostat at room temperature for 1-2 min to completely dry. Store sections in the cryostat no more than 30 min before further processing.

I t is not my field but i read for you the follow dection , i hope it will help you read more on this paer "Muscle fiber activity as a function of speed and gait"

The portion is mounted on a specimen holder and frozen in 2-methylbutane cooled with liquid N,. Serial sections were subsequently cut from this sample in a cryostat at -2O”C, mounted on coverslips, and stained for diphosphopyridine nucleotide- diaphorase (DPNH-diaphorase) and myofibrillar adenosine triphosphatase (ATPase) activities, and for glycogen with the periodic acid-SchifYs (PAS)

Stain.

Dr A.Ouerdane from Namur Belgium

Read section in line 1

Please allow the cryoprotected samlpes to thaw at room temperature then wash off sucrose with PBS before embedding in the OCT. You would get good quality cryosections. Try and report back. Dr. CS Rayat ([email protected]).

Ok i share your point of view Chran Rayat i vote for your response by yes.

I am wondering why you have fixed the tissue to begin with. Cryo sectioning is ok to section fresh-frozen tissue. That said, it sounds like your tissue is pulling out of the OCT. Try several things; adjust the knife placement to a very sharp area, put block into stub holder in the locked hi-position, carefully trim block into pyramidal-shape used for paraffin-sectioning, lastly freeze with freon spray. I believe that you need to get more even/confluent temperature btw tissue and surrounding OCT. Hope this helps.

There are few possibilities specially when you are not getting proper sections, I will try to narrate few and you can check

1. Type of tissue you are using and with this fixation time is very important as you named only fixative not the fixation time, so check with with your fixation time, over fixation can lead to many of sectioning problems specially tissue become powdery. After fixation wash your tissue with buffer for several times.

2. Check with you razor, observe the razor edge under the microscope if you find nick ans scar on that sharp it. Check with adjustment of antiroll plate.

3. Keep an eye on cryostat chamber temperature with both the things are fine which I described above then temperature is the only reason your tissue is getting over cooled and if this the case rub your palms for few seconds and put them near to the tissue and try to section.

OK ,Sunil i agree with this technique, it is useful for our Rosa especially the need to keep one eye on cryostat chamber temperature which be carefully controlled.

Dr A.Ouerdane

In my experience I think the problem is in the way you have fixed the samples....at this point I can suggest you to try to avoid the cryoprotection of the samples to see if OCT can penetrate better into them or to try the technique suggest by Charan Rayat.

For next time I suggest you to reduce the time of fixation.

I agree with Dr. CR Rayat's suggestions and I also think you should try out a few different temperatures within the cryostat.

Yes Amanda, it was also my point of view; Rayat idea and the technique proposed by D.S Sunil are the two advises to our Rosa.

thanks everyone for the suggestions, it is the first time I wrote on the blog and I didn't expect all this answers :).

I have few things to reply for some of you to clarify .The samples are not floating away my glass slide, not yet rich that step. I loose the sample while I'm cutting it. I think the problem maybe is in the fixation..or some of the step between it and the cut at the cryostate.

- Dr.Charan Rayat I already try what you suggested but didn’t work.

-Doria I did fixed the samples 1st because I need to collect different stages and I didn’t have time to work on all of it, second I was following a paper where they suggest this protocol. but unluckly for me didn't work.

-Sunil Which buffer you mean PBS? I do wash the sample with PBS before and after the cryoprotection. I read that in FAA the samples can stay for months. I do check the temperature of the cryostate and I left the sample for 1hr before cutting it. I change the blade the thikness and the speed of the cut at the cryostate.

More or less I already try all the suggestion you all gave me I will only try what Alessandra suggest to fix without the step in sucrose and keep an eye on the blade

I can't work on fresh tissue because right now I don't have it available. they are local ecotype of tomate only harvest in the early summer.

thanks

Alessandra Gastaldello

should I wash the samples in PBS before using OCT or just like that?

thanks

Yes, I think should be better to wash the samples with PBS, at least two times for 5 minutes, better if in mild agitation.

Hope you can fix the problem!

Thanks!

Rosa had you try to take the advises given by all the eminent scientist ,? if yes you can give us what is the news.We are all interested if your problem is solved and how?

I never even heard about someone sectioning tomatoes (=vegetables) but are you sure you need CRYOsectioning for that? For me it seems more probable to cut fixed vegetables with a high aqueous part at a temperatur around 0°C where they might be more elastic without cracking and without fast destructions of proteins.

During cutting of the sections in cryotome temperature is very essential. If temperature is too high means it should be between 21-24 degree centigrade. Otherwise section will not come properly.

If you use tissue sections for immunostaining and cryosectioning is difficult, alternative way of tissue processing is paraffin embediing.

See: Proliferative activity of human thyroid tumors evaluated by proliferating cell nuclear antigen/cyclin immunohistochemical studies.

Shimizu T, Usuda N, Yamanda T, Sugenoya A, Iida F.Cancer. 1993 May 1;71(9):2807-12.

Hi Rosa,

I have only worked with animal tissue. However, the mix of alcohol in your fixative is preventing your tissue from freezing. Can you fix in 4% paraformaldehyde for example or regular 10% NBF?

I am sure that if you avoid the alcohol and use sucrose to cryoprotect you will be in business. If all of your samples are already fixed this way, you might try to wash one sample in numerous changes of your favorite buffer and hope that the alcohol will be washed out. Freeze and cut again.

Good luck!

Paraformaldehye is cross linking fixative and reduce RNA recovery....

Paraffin embedding is more prone to RNA degradation

I replaced the membrane slides with superfrost positively charged slides, and lossing of sectioning is minimized.

L"m not familiar with vegetable tissues, however I don't think that fixation is the cause of your problems. Cryocut sectioning works also on non fixed tissues. Other causes that are mostly cited could lead to this sectioning problem.

- Try to cut without using anti-roll

- Keep as possible your sample at the same temperature of cryostat. In general, keep it at least 1 h in the cryostat before sectioning

- the inclination angle of the blade should be optimal

'Ensure that the quality of OCT is as good as possible. You can also try to cut your tissue without using OCT. Why not?

- 

HELP!!!! my TA muscle sections have been left out of the -80°C overnight by mistake. WHAT should I do? are they still good???

@ Angelica: Dry sections on slides are quite stable. One night at room temperature should not harm them too much, depending on what you want to do with them (I would not look for DNA/RNA anymore). Make sure that you have a control when staining them.