I have some samples ready to be processed for paraffin embedding, but when I cut the decalcified joints into 3mm thickness sections to fit into the paraffin cassettes, the tissues tend to separate from one another. Any ideas how to keep the tissues together so that I can prevent them from separating in the paraffin block before sectioning? Edit: I have heard that using mesh, like from sanitized stockings, might work, but I have never done any kind of histochemistry before so I am not sure how this would work.
Hi Laura,
This problem could be related with your dehydration and clearing processes. If your tissue sample was not dehydrated enough, xylene could not get into tissue sample completely (xylene cant mix with water), as well as the heated paraffin. When the tissue was not fixed well by the paraffin, they will be separated when you sectioned them. Hope this will help. All the best.
Hi Laura,
You could try embedding the joints in some gelatine before the dehydration and then on cutting the tissues they will hold together. The gelatine will just go through normal dehydation and into paraffin as usual. The gelatin can be trimmed to the size you want surrounding the joints.
Hope this helps.
I think using double embedding with gelatin before paraffinaization is good idea
Or put it in whole, into a mega-cassette. However when it comes to the paraffin embedding I swop the cassette for a standard one as they are a better fit in the microtome chuck.
Great, good advice. I will definitely consider these options when I run into this issue. Thank you!
I used to section the fixed knee joint/femur with a fine diamond lapidary saw. From a range of species large and small including the rat. So that it fitted into a standard cassette. Then I would decalcify it/them en mass, in a large container with a magnetic stirrer gently agitating the fluid. Checking the 10% formic/5%formalin decal fluid with Arnim's test. When fully decalcified a simple case of loading onto a processor and embedding. With no handling in between...
Hello Laura
Is the usage of a cassette absolutely necessary? If you make a larger block, you can embed the whole joint (if it's mouse or rat, what is it, really?). In our lab, we use an old-school method for this by taking two L-shaped pieces of metal and putting them together to create an approximately 2.5x1.5x1.5 cm mold in which we pour liquid paraffin and then put the prepared bone/joint inside using tweezers. You get a nice paraffin block that can be fitted on different microtomes. You can use silicone molds too (for ice-cubes, pralines and similar stuff, just make sure it can withstand the paraffin temperature).
Hope that helps.
An additional trick is to leave some of the adnexal tissue attached, such as the muscle on the bone, it helps the joint "key" into the paraffin wax. And prevents it from shelling out of the block when you come to sectioning on the microtome.
The L shaped moulds on the comment above, are Leukhardt moulds, it all depends on what type of chuck you have fitted to your microtome. The common chuck is for the "Tissue Tek" type of cassette. If you are sectioning human knee joints you can get "Super Mega cassettes" (who makes these names up! ;-) the matching extra large S/S moulds and again you need the matching microtome chuck...
Good points by Peter.
Maybe, with some manual work, even this paraffin block made without a cassette can be fitted in a cassette you have. Depends on your rig and experience. But I think there are ways, and from my experience, it's better to embed the whole thing and then cut. So, I'd look for a workaround instead of cutting it to 3mm...
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology