First, make sure you are washing all of the PFA off (using PBS) before putting it in the sucrose. Then, I would suggest using a higher percentage sucrose solution, going up to 35-35% and (as you are already doing) waiting for it to sink. This may better protect the tissue.

Next, look at your blotting. You are using Whatman filter paper? Too wet and you get the Leidenfrost phenomenon (poor freezing), too dry and you will also get artifacts. 

What temperature are you storing your OCT embedded samples? It should be at -20 and not -80. If stored at -80 then the OCT may change and cause the blade to wobble and you will get uneven sections.

Artifacts are also caused by slow freezing. As you probably know, fast freeze = small ice crystals and less tissue damage. You have tried the methylibutane-liquid nitrogen method - an option is to freeze the sample in isopentane that has been precooled to liquid nitrogen temperature, which will not cause bubbles and can shorten the freezing time. One of our protocols calls for a slower freezing of the OCT capsule (especially for tissues with little intrinsic strength) by either 1) immersing the bottom first, allowing it to freeze, and then submerging the whole capsule or 2) placing a puddle of OCT on a flat piece of dry ice and as the OCT begins to freeze, sliding the piece of tissue into the solution then allowing it to freeze completely.

If the pre-cutting procedures aren't the problem, then look at how the sections themselves are cut and handled. Be sure to check your blades. Nicked blades cause split sections. Some of our protocols call for floating the cryosections in cold buffer (PBS) to be floated onto the slide. Also, make sure there are no bubbles under the sections. And we will also use either Poly-L-lysine-coated, silanized, or 1% gelatin coated slides to help with adhesion.

I mention several protocols because it really depends on the tissue you are working with. Most have to be optimized for each experiment.

I am interested to hear other suggestions on the matter as well. 

Good luck!

Dear Sandra,

Have you tried perfusing the heart with the fixative (4% PFA)? It may be that your fixation is not very effective, just immersing in fixative. The fixative may take a long time to penetrate the tissue and reach the deeper cells, which may become necrotic by the time they are fixed. I work usually with other tissues and therefore I perform intracardiac perfusion of the whole animal prior to tissue collection and that way all the tissues are fixed within a few minutes of perfusion. See my thesis on RG for the details of method.

best wishes,

Refik

I had same problem with brain tissue. I found out that the problem was that I used to put the organ first in OCT and then freeze it.

For some reason I don't know, I got "bubbly" tissue sections and I had to trash all of them, just because performing immunohistochemistry afterwards proved to be just useless.

My final suggestion is to freeze the organ as it is, after sucrose protocol. Only when you are about to section, embed the tissue in OCT until it freezes (usually 20 minutes or so in a regular cryostat) and then start slicing.

First, make sure you are washing all of the PFA off (using PBS) before putting it in the sucrose. Then, I would suggest using a higher percentage sucrose solution, going up to 35-35% and (as you are already doing) waiting for it to sink. This may better protect the tissue.

Next, look at your blotting. You are using Whatman filter paper? Too wet and you get the Leidenfrost phenomenon (poor freezing), too dry and you will also get artifacts. 

What temperature are you storing your OCT embedded samples? It should be at -20 and not -80. If stored at -80 then the OCT may change and cause the blade to wobble and you will get uneven sections.

Artifacts are also caused by slow freezing. As you probably know, fast freeze = small ice crystals and less tissue damage. You have tried the methylibutane-liquid nitrogen method - an option is to freeze the sample in isopentane that has been precooled to liquid nitrogen temperature, which will not cause bubbles and can shorten the freezing time. One of our protocols calls for a slower freezing of the OCT capsule (especially for tissues with little intrinsic strength) by either 1) immersing the bottom first, allowing it to freeze, and then submerging the whole capsule or 2) placing a puddle of OCT on a flat piece of dry ice and as the OCT begins to freeze, sliding the piece of tissue into the solution then allowing it to freeze completely.

If the pre-cutting procedures aren't the problem, then look at how the sections themselves are cut and handled. Be sure to check your blades. Nicked blades cause split sections. Some of our protocols call for floating the cryosections in cold buffer (PBS) to be floated onto the slide. Also, make sure there are no bubbles under the sections. And we will also use either Poly-L-lysine-coated, silanized, or 1% gelatin coated slides to help with adhesion.

I mention several protocols because it really depends on the tissue you are working with. Most have to be optimized for each experiment.

I am interested to hear other suggestions on the matter as well. 

Good luck!

Dear Mr. Mohammed,  Mr Refik, Mr. Placido, and Ms Mellisa,

Thank you so much for all suggestion, I really appreciate it.

Let me try one by one, and see the result.

Actually I did not do the intracardiac perfusion, since I am a beginner in the in vivo model. If I know it before,  I would really try it.

I use a simple blotting tissue paper (Kim paper) to make the heart dry. After freezing, usually I put n -80 celcius overnight, and put in -20 celcius 30 minutes prior the slicing.

I will keep all the suggestion and update the progress.

Here I also attach my previous condition of my slide, it was so bad.

Best regards,

Sandra

I think the problem in fixation step. 

First of all: There should be no big difference between 4% PFA and 10% formalin, since 100% formalin is the typical name for a 37-40% PFA solution. Diluting it to 10% formalin gives you a final concentration of 3,7-4,0% PFA. The main difference is that commercial formalin solutions are normally stabilized with up to 10% methanol - which can be good or might have a large impact on your experiment.

Also 2-methylbutane and isopentan are the same thing...

Second: Muscle tissue (heart is a big muscle) is quite sensitive to osmotic changes, so be careful with PBS and physiological saline before fixation.

Third: Sometimes sections crack on the slide if the slide is not clean and regularly coated, although it looks a bit differently from your picture. Never the less, try to get high quality pre-coated slides, putting only one section to the slide.

Forth: For muscle tissue a substance called "Tragacanth" is often recommended instead of O.C.T. medium. For my experience it did not change much but you might look for protocols and try it. Have some patience while dissolving it...

Fifth: Have you tried to use unfixed frozen sections without any washing and fixation steps before freezing the tissue (in nitrogen cooled methylbutane)? The morphology might be a bit worse than with fixed and embedded tissue but better than what you actually get. And if you want to use the sections for immuno stains afterwards it makes things easier since you have kind of a gold standard how the antibodies should look like when trying to find the right retrieval procedure.

Sixth: If there's not experimental need for sectioning the heart as whole organ, try to make smaller pieces before freezing. This will help quick freezing of the tissue.

Seventh: Do not go directely into high concentration sucrose solutions after fixation, start with 10% until it sinks (might be quite fast), go to 20%, then 30% or 35%.

I think your problem is a combination of liquids (washing, fixing and cryoprotection) before freezing and bad quality slides.

Good luck.

We do not fix the heart at all. Just place in PBS for 30min, gently tease out any blood clots and dry excess PBS, then place in OCT in an embedding mold for 30-45min. Then, freeze for 20-30min in dry ice cooled isopentane.

Have you tried a different set of slides? My guess is that the tissue is not adhering to the slides properly, and therefore the morphology is distorted when the sections dry on and pull away from the slide. You could try coating your own slides (a pain) or just a different batch of commercial slides. We use Fisher Colorfrost Plus slides. It also helps to warm the slides before adhering the sections to them.

Good luck!

 Hi Sandra,

Did you fix this drying problem yet? I'm having pretty much the same kind of trouble recently. 

Great ,  A very nice study conducted by great scientific approach. I will be happy to share my suggestions.