Hi there!

I don't know how mouse testis tissue behaves. We're working with brain tissue (both human and rodent) and some times we have also this problem as it is a very delicate tissue. 

We also work with flash-frozen tissue (liquid nitrogen freezing and then keeping tissue at -80ºC embedded in OCT) for laser-microdissection. What it is also important is the temperature at which you're cutting the tissue in the cryostat (for brain, a -20ºC is preferrable).

After having the tissue slices in the PEN-membrane slides we use to microdissect, we fix the tissue with cold acetone prior to do the staining and dehydration of the sample.

Hope it helps!

Have you done a sucrose gradient before freezing your tissue sample?

How do you perform your freezing (liquid nitrogen? -80°C?-20°C? ) OCT embending?

Freezing must be done in a very fast way and to preserve your tissue integrity you need to dehydrate your tissue to avoid H2O crystal formation in your tissue. In addition using embending that surround your tissue will help to keep the original structure before freezing.

Hi there!

I don't know how mouse testis tissue behaves. We're working with brain tissue (both human and rodent) and some times we have also this problem as it is a very delicate tissue. 

We also work with flash-frozen tissue (liquid nitrogen freezing and then keeping tissue at -80ºC embedded in OCT) for laser-microdissection. What it is also important is the temperature at which you're cutting the tissue in the cryostat (for brain, a -20ºC is preferrable).

After having the tissue slices in the PEN-membrane slides we use to microdissect, we fix the tissue with cold acetone prior to do the staining and dehydration of the sample.

Hope it helps!

Hi,

when we snap freeze our skeletal muscle biopsies, we freeze them in isopentane cooled to almost freezing in liquid nitrogen. We have found this gives us better tissue morphology rather than freezing directly in liquid nitrogen. Also, as mentioned above, the temperature in the cryostat is important. Good luck!

It might be helpful to get a picture of you stain to see what might be the problem.

Since your sample is already frozen, keep it frozen, don't try to put in any cryo-protection solution afterwards. Try to find a HE-staining protocol especially for cryosections. Do not let the sections dry out during staining. You might also try cutting sections of different thickness (e.g. 5µm, 10µm).

Next time you prepare testis samples, think about freezing procedures.

Frozen section is always not clear epically with old specimen . So my suggestion is if you have a chance take a small piece from your tissue and make paraffin block section.

Hi,

Thank you all for your suggestions and wishes, I appreciate it. I tried the snap freezing in liquid nitrogen and the sucrose treatment but that didn't help to improve the quality, I guess because my tissues are already frozen! I think I may try doing the sectioning without cryo-protection solution or doing paraffin sections as Vivica and Mansor suggested.

Hi

As the tissue is already frozen,one important thing to maintain integrity is to pr event crystal formation by dehydrating the tissue meticulously.you have snap freezed tissue  in liquid nitrogen,then there should be no problem.

I think there may be more than one effect to be mindful of. First, endogenous proteases may destroy architecture in the absence of fixation or getting the tissues cold as soon as possible, but that varies by tissue. It is most likely that the distortion you see is due to lack of cryopreservation (e.g. graded sucrose) prior to freezing. Ice crystals, as discussed, are likely to form with any freezing so the sucrose would need to be infused prior to dropping the temp. I would think that any standard fixation will not change that outcome. I am not familiar with testis, but if you must work with the current samples, you might try to section at slightly increased thickness to give you  more to look at. The compromise will be in resolution of structures. A standard H&E should work, but you could also use other vital dyes (e.g. PAS, toluidine blue) to reveal different/more structures depending on what it is you are trying to see. Best of luck!

Thank you very much for your valuable comments. I will consider what you suggested in my experiments.

Fixinga short time (10 min?)  with buffered formaldehyde after collection...then do PCR or other molecular test.

There are many books (Hayat) and published papers about such procedure if you look in google...or pubmed. I am surprised you have stained with H&E where H can intercalate and obviously interfere with your search.

The sucrose gradient has worked the best unfortunately in formalin fixed material for us.  IF you can use formalin, give that a try.  Otherwise the snap freeze in LN2 is best bet with unfixed material.  First I suggest to cut the testis in half before freezing, and yes I know in mice they are tiny little fellows. (Do this before sucrose gradient too to ensure penetration.) The capsule can restrict expansion sometimes and make a mess of the structure at least when I have processed for paraffin so perhaps it would be the same for this.  If I think of something else I will let you know!

Thanks all for your suggestions I do appreciate that.

You do not want to snap-freeze in liquid nitrogen, as snap-freezing causes ice crystals to form and that destroys the cellular integrity.  The best way (in addition to Dr. Ashraf Imam's suggestion on surrounding the tissue with OCT compound, which is what we do in the clinical setting of an intraoperative frozen section) is to use a heat extractor, as this provides an even extraction of the heat to cool the tissue, resulting in the least amount of tissue distortion.  You can use a cryomold which is a block of chrome-coated metal with recessed wells to hold the tissue and OCT compound.  This acts as the heat-extractor and provides for an even plane of section; further you can better control the orientation of the tissue that you cut with these wells.  There are other devices that you can use to make almost "permanent section" quality that is used in the clinical setting; however, they may also interfere with anything that you intend to do for immunostains or other studies on the frozen sections.

Liquid nitrogen can be great if isopentane is used to immerse the tissue to avoid ice crystals forming...

Isopentane in a device designed for rapid cooling also can reduce ice crystal formation; however, the odor emitted from the Isopentane sent some of our employees and the reference lab employees home sick, so we discontinued using it about 10 years ago.

Thank you all. Wesley I think you made a good point that I should consider. I will give it a try.