I need to visualize the CMFDA/PI stained cells within the scaffold, but dehidratation steps needed for paraffin embedding of the samples partly erose my PLGA scaffold. I read in many papers that they use paraffin embedding with this kind of scaffolds, but no one says nothing about the protocol used. Another way could be to use OCT instead of paraffin, avoiding dehidratation steps. Any suggestion?
Thank you in advance!
yes I would suggest use cryo sectioning, Use OCT medium and liquid nitrogen,
Cryo work for me I used porous chitosan samples with hydroxyapatite
I agree with the Cryostat, the problem with the paraffin embedding is that you will most probably loose the PLGA or end with deformed scaffold when you use organic solvents for washing.
I suggest using cryosectioning with tape transfer of sections. It seems to hold up the scaffold structure better.
Thank you everybody! I decided to use cryo preservation of the samples, as you suggested!
cyropreserve seems to be good protocol to follow. in addition, i have freeze my scaffold in liquid nitrogen first, then follow by cyropreserve.
Would suggest still to not stain your slides all at one because sometimes scaffolds detached from the slides. so just check with your usual staining protocol that everything stay still.
I am sorry, I have another question now. I am preparing the cryosections of my PLGA scaffolds (in OCT), but they do not adhere to the slide and, due to their porosity, they are brittle. How can I preserve their integrity? Do you have a good protocol, from the cryopreservation to the slides preparation, and so on? Thank you very much in advance!
Nancy contact Anna Pego she is on Research gate and worked with scaffolds.
Nancy you should try different temperature of cut. Furthermore try to use super frost or superfrost plus. That should help. There should be also Lysine coated glass slides but with my polymer they were not working.
Furthermore what is the thickness of the slices you are cutting?
Dear Annunziata,
I used OCT compound to embed my hydrogels. To cut my hydrogel into slices, I used LEICA cryotome (https://drp8p5tqcb2p5.cloudfront.net/fileadmin/downloads_lbs/Leica%20CM1100/User%20Manuals/Leica_CM1100_IFU_1v3B_en.pdf).
First, I made alumunium mold from an alumunium foil, then the hydrogel was put on the mold and added the OCT compound. Freeze the mixture at -20 degree C. On the top of specimen disk (see the instruction manual), I put a little amount of OCT compound put at -20 C.
Attach the frozen hydrogel (remove the alumunium foil) on the top of frozen OCT on the disk, and cut it into the desired thickness using a cryotome. The slices were then fixed on glass slides coated with adhesive silane. After drying and washing the slices, you can stain them.
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