Maria: In my experience thawing and refreezing causes disintegration of the tissue. Has the brain been fixed or cryoprotected in sucrose prior to freezing?

Karen Bentley

Hi Karen- It was fixed in 4% PFA but it was not cryoprotected. At this point the block is so badly deformed that are considering it almost a lost. How did you thawthe tissue that disintegrated, fast/slow, in a bath or transferring to warmer freezer ? Thanks,

Maria

Maria:

I put it in a -20C freezer for several hours then allowed to thaw at 4C. It became quite soft. Much softer than original tissue. When refrozen and sectioned we saw areas of cellular disintegration leaving gaps in the tissue.

Is it possible to thaw at 4C in fixative such as 10% buffered formalin?

If the tissue has not been cryoprotected with a sucrose solution (I use 30% in PBS) there will be a lot of freezing artifacts. I don't know that thawing fast or slow will make a difference in this case. I like the idea of thawing it in the fixative (I would keep with the 4% PFA), then cryoprotect and re-freeze. It's worth a shot since the tissue is considered a loss at this point.

Depends on what you plan do with the tissue later. If you are interested in morphology, it will be very poor quality regardless of what you do. If you can use it as is, I would recommend that.

if u need to look at some antigens, I thik it will be OK, but for morphology, cause ICE crystals may cause some damage to cells so I dont expect you may get good histological sections.

Dear Maria Navas-Moreno , it would be helpful - I think - if you could tell the community here about the size of the brain specimens you froze after 4% PFA-fixation and, if this is possible, also which area of brain and which direction / sectioning plane was chosen. As you said that the most convexly bent specimen was "embedded" into gelatin the answering for area and dissection plane (longest axis) would be interesting since bending or convex deformation has to do perhaps with certain brain structures(e.g. cellular -fibrous) OR - at least - with the gelatin stuff itself (perhaps other physical parameters of contraction/constriction of brain tissue vs. solid and then frozen gelatine). Best regards

Dear Maria,

you put the tissue block from -80°C freezer in a -20°C freezer. Immerse the tissue in room temperature Iso-pentane until the tissue is completely thawed, agitate the tissue block in the iso-pentane and refreeze the block as you did earlier in a new solution of iso-pentane. Good luck, let me know what you decided and what the outcome of your experiment is.

Dear Wolfgang,

The specimen is a block of the most anterior part of the brain. The gelatin is on the anterior part of the block and what got badly deformed is the posterior face of the block which is exposed (not in gelatin). The tissue is about 2 cm long AP and 4 cm left to right. We are going to section (coronal) and do immunohistochemistry. The problem with the deformation is that we don't think we will be able to recognized smaller anatomical features and thus have a hard time interpreting results.

I think what cause the deformation was some 20% glycerol that got trapped in between the gelatin and the tissue, at least for this case. Before, the convex feature might have been cause by a difference in the thermal properties of various parts of the tissue as you suggested.

Best regards