Hi all,
We are using for some time now the Kawamoto film method to section limbs which contain calcified bone at different ages. However, we have noticed that when taking the section out of the cryo for further staining, the soft tissue around the bone begins to shrink rapidly and within 1-2 minutes, the tissue is about half of its original width and the morphology of the tissue is damaged. This happens when using both a regular OCT and the SCEM that is recommended for use with the tape. This happens both in fixed and fresh tissue, and also in tissue that was dehydrated with 30% sucrose, although shrinking is somewhat slower.
If we keep that tape with the section frozen this does not happen, but if you transfer a frozen sticker directly to PBS for staining than there is a greater chance that the section will come off the sticker.
Any ideas as to how to solve this??
Dear Neta,
I am also using the Kawamoto cryotape method and have a similar problem. I am working on human undecalcified osteochondral tissue (cartilage-on-bone). The cartilage pericellular matrix shrinks, leading to the loss of whole group of chondrocytes within a lacuna, leaving big holes on the stained sections. I tried different snap-freezing methods (liquid nitrogen, isopentane, dry ice), different chamber temperature (from -20 to -50), different section thickness (from 3um to 30 um), different post-section fixation protocols, still do not have a solution.
Regarding the sections coming off the sticker, I think you just have to press harder when attaching the film to the tissue block, which worked for me.
Have you solved the shrinkage problem since you posted this question here?
Thanks!
Dear Yunfei Li,
I have not solved it but with further experiments, we have found that:
1. Prolonged drying of the tissue in the chemical hood greatly helps maintaining the tissue on the sticker, just like with regular frozen sections.
2. Adhering the stickers to the slides (we use Chitosan glue) before immunostaining is a must for uniform application of the antibody
3. In a few independent instances, although the soft tissue shrank dramatically after sectioning it regained its original morphology following washes in PBS and other water based stainings. However, staining for H&E or other stains which require dehydration and mounting in Xylen based glue always resulted in a shrunk tissue, and to this I have found no solution yet. An example for this can be seen in the attached file- these are two consecutive sections taken with the Kawamoto film method, one rehydrated and stained with DAPI; the other dehydrated and stained for H&E.
Hope this helps,
Best,
Neta Felsenthal.
This is Achilles tendon attachment to the cartilage of a neonate mouse. The tissue was fixed in 4% PFA for 3hrs in 4C, washed in PBS, changed to 30% sucrose and then embedded in regular OCT.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Sectioning