Hi All,
Currently I am working in tissue cryosectioning with CMC (4% CMC was prepared, preparation for MALDI imaging analysis). However, I noticed that the CMC is lack of structural integrity and unable to embed the tissue well. While sectioning, both tissue and the CMC matrix are very brittle and disintegrate easily, which made me unable to fish out the tissue with intact structure. May I know does anyone facing the similar problem and how should I improved? Many thanks!
Hi, I've used it with bone tissues, here is what I did:
1) Tissues were fixed and decalcified in 10% trichloroacetic acid (TCA) at 4C for 24h.
2) Tissues were subsequently cryoprotected in 30% w/v sucrose in 1x PBS:
2.1) Place each tissue in a bijoux filled with 30% sucrose in 1x PBS
2.2) Initially tissue will float, incubate with gentle stirring at RT until tissues sink.
NB: This step is to prevent large crystal formation during the embedding step; this ensures better quality sections.
3) Embedded in 3% w/v carboxymethylcellulose (CMC), and store at -70°C prior to sectioning (4% is probably also fine)
3.1) Prepare a dry ice/EtOH slurry. Add dry ice to an inch from the top of a small styrofoam box and pour ethanol slowly. It will bubble and steam lots. Ensure you cover the dry ice with a very thin layer of EtOH and leave to equilibrate (The boiling will stop).
3.2) In the meantime, prepare plastic moulds for embedding that fit the size of your tissue. Pre-label the moulds.
3.3) Place tissue inside, adjust orientation and cover with 3% CMC. I use a pasteur pipette to pour it over my tissues. Re-adjust orientation if needed. Using fixed samples allows you to let the CMC penetrate as much as possible (leave for a few min in the CMC); before placing tissue in the mould you can tap it on a blue roll to dry it as much as possible and to allow better CMC penetration.
3.4) Once dry ice/EtOH slurry has equilibrated, float the mould on top of it and take care not to mix the CMC and EtOH.
3.5) The CMC will fully turn white when it has frozen. After that happens. wipe the mould with a piece of blue roll, and make sure if EtOH has come in contact with the CMC to take extra care to wipe any leftover EtOH.
3.6) Place the embedded block on dry ice and store at -70°C prior to sectioning.
Hope this helps and sorry for the late response!
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