Tissue Removal from Hydrogel

gjonesgjones Posts: 12
edited July 2013 in Hydrogel Embedding
Hi All,

We're having issues with removing the sample from the gel.
The gel is extremely rubbery and as a result there is often a significant amount of gel left behind on the sample.
Is anyone else experiencing this or could shed some light onto the reason as to why this would be occurring?

Thank you!

Comments

  • alexalex Posts: 2
    Our hydrogel is also very rubbery and more difficult to cut than it looks like. In some cases it more tougher than tissue. I've torn apart a spleen when I tried to remove it from the embedding and trim the excess. With other tissue situation a bit better.
  • I'm pretty sure that tough gel is indicative of good polymerization. Personally I no longer attempt to cut each brain out of the gel. Now I just cup the tips of my fingers around the brain, and with the other hand, rip the gel off. The gel seems to normally break at or near the tissue/gel interface, which is perfect. I have noticed that if the perfusion was poor, or the tissue was not well embedded, this method may damage the sample, because the sample will be relatively soft. I imagine that softer tissues (the spleen mentioned above) might not fare so well with this technique.
  • LauraLynchLauraLynch Posts: 31
    I've found that, once most of the surrounding gel is removed, gently rolling the tissue on a paper towel or Kimwipe helps get rid of the rest.
  • houhou Posts: 2
    I tried wrapping up the brain with one layer of gauze before polymerization and found that this could help remove excessive gel around tissue.
  • I agree that using your fingers to break off the large chunk of hydrogel from your sample is easiest. I'd suggest using your fingers (use a fresh pair of dry gloves) to gently rub off the remaining hydrogel from the surface of the tissue or gently rub it off with a Kimwipe.
  • Also, for anyone using samples that are too fragile or small to rub off the excess hydrogel, you can replace the bis-acrylamide crosslinker in the hydrogel solution with water. This will prevent the liquid outside of the tissue from crosslinking during polymerization while a hydrogel will still form within the tissue.
  • gjonesgjones Posts: 12
    What exactly is the advantage in using bis-acrylamide then?
  • The bis-acrylamide directly crosslinks the acrylamide polymer chains. This helps to strengthen the hydrogel, especially in void areas where there are less biomolecules present (the biomolecules also indirectly crosslink the acrylamide chains by reacting with formaldehyde which then reacts with acrylamide).

    So, the bis makes the hydrogel stronger so it can be easily handled and so that larger samples especially don't fall apart. This is less of a worry when you already have a small, fragile sample, although generally incorporating bis is always better for hydrogel integrity.
  • Hi,
    It seems like the consistency of the gel differs between groups using CLARITY. I was wondering what the consistency is supposed to be like? After polymerization, my gel is more like pudding/flan.
    I just put the 50ml falcon in a 37C waterbath after I have replaced oxygen by nitrogen. Is the tube supposed to be on a shaker while polymerizes?

    Thx
  • FabioFabio Posts: 61
    Hi, my hydrogel has also a flan consistency. Its also kind of sticky. Normally I turn the falcon tube up-side down after polymerization and if nothing moves is ready.
  • yetitiyetiti Posts: 7
    pudding/flan sounds about right. You do not want your gel to be too rigid--ie, do not leave polymerization in 37c for more than 7-8hours or o/n. otherwise the clearing would be very difficult.
  • aysegulaysegul Posts: 33
    Does anyone have any suggestions to remove excess polymer on the sample after hydrogel embedding? We are having a little problem. Polymerization on the sample is very sticky. When dealing with my finger to remove excess gel it doesn't work. But i'm sure that tough gel is indicative of good polymerization. Does paper towel or Kimwipe helps get rid of the rest ?
  • AdamAdam Posts: 31
    Yes, just rub the sample with a paper towel, or roll the sample around on it, both work well.
  • aysegulaysegul Posts: 33
    Excess gel sticks on paper but not leave on the sample
  • dgrobertsdgroberts Posts: 16
    @aysegul You may have overpolymerized, but try a Kimwipe (not a paper towel) and see how that goes.
  • Hi @aysegul. I agree with the post(s) above. You actually don't want the gel surrounding your sample to be too tough because the resulting hydrogel in your tissue is likely to be over-polymerized and crosslinked, such that it will not be porous enough for good clearing and, later on, staining. The gel that you get after 3 hours of polymerization should be solid (i.e. no flow), but very flexible and bouncy. It should easily rub off from the sample with either a Kimwipe or your fingers.
  • aysegulaysegul Posts: 33
    Thanks for everybody for suggestions, @Kristin_Engberg it is very useful information for other trials. This is just preliminary trial.
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