Hey Jooeun,

Send your complete fixation protocol please. It could be because of the earlier steps as well and not necessarily the embedding step.

Cheers,

1. Fixing

2 % PFA, 0.1 % Glutaraldehyde overnight

2. Dehydration

50% EtOH/50% water   30 min

70% EtOH/30% water 30 min *

80% EtOH/20% Water  30 min

2:1 LR White resin to 70% EtOH 1 hour**

100% LR White resin 1 hour

100% LR White resin overnight

100% LR White resin 1 hour

100% LR White resin 1 hour

3. Polymerization

Replace 100% LR White resin and remove air, then add accelerator and cure at 55 degree for 30 min

Thank you for answering me

Hey Jooeun,

To me your GA concentration is very low. I used to do 2.5% when I was fixing samples for EM. A lot of the protocols I read do 2/2.5% GA and no use of PFA. Do you have a specific reason to use the PFA? I would recommend dropping the PFA if you are aiming at solely structural work.

Plant samples are tricky to fix, one thing to do but it seems to me that you are going this way also is to make the embedding times longer. So instead of doing 1 hour, make these steps longer. I don't have experience with LR white. Maybe try Spurr resin?

Bottom line, in order of priority, I would first start increasing that GA concentration, maintaining all the other stuff the same and seeing if this works. Then you can lenghten the embedding times and then maybe test other resins?

Perhaps using acetone for dehydration steps also?

I hope this is not too messy and it helps.

Cheers,