Hi, we’ve been clearing the brain tissue for three days but had several problems along the process.
Details of our setup are as follows:
solution volume was up to 9L (actually we made 10L of solution but the water bath could hold only 9L.),
miliQ 5 micron filter was used (the filter tank was taken from ice maker that is not in use anymore),
voltage was about 30V (the amperage fluctuated from about 0.7 to 1.4A), and
temperature was about 37℃.
You can check our setup here: https://docs.google.com/file/d/0B5smZKUGCA3ZeE5xY3RXRkt2Y2s/edit?usp=sharing https://docs.google.com/file/d/0B5smZKUGCA3ZRWEtMU1HMWRjeEU/edit?usp=sharing https://docs.google.com/file/d/0B5smZKUGCA3ZbzRjeTFadC0tSTQ/edit?usp=sharing
Tissue clearing was run for last 3 days but we couldn't find any “cleared tissue” there.
You can check our tissue which went through 93 hours(3 days + 21hours) of tissue clearing here: https://docs.google.com/file/d/0B5smZKUGCA3ZMWxLei1QRWV5VkE/edit?usp=sharing https://docs.google.com/file/d/0B5smZKUGCA3ZX0lLQkNuSkMtaHM/edit?usp=sharing https://docs.google.com/file/d/0B5smZKUGCA3ZU2V4ZHpqVmYxeUU/edit?usp=sharing
Last time we tried clearing brain tissue with 3L of clearing solution, only to find yellow and opaque tissue at the end.
I'm afraid we may find yellow, opaque tissue again this time...TT
So we happened to think of several problems of our setup.
1. Filtering system may not work properly so the solution is saturated with lipids.
2. Evaporation of solution somehow made it saturated with lipids.
3. Combination of factor 1 and 2 is somehow blocking lipids to be moved along the flow.
4. Voltage and temperature is not enough to clear the lipid in brain tissue within 3 days.
At this point, I want to ask you several questions regarding our setup:
1. Are there any problems with our setup? (e.g. filter pore size or voltage, amperage, temperature)
2. Do you think our filter works well?
3. Is the contamination level of our solution normal?