Need some help on our ETC setup....

Hyewon

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?

liz_kritzer

What kind of pump are you using to circulate your fluid? It could be that the circulation is poor due to the pump and not your filtration setup. Also, is there a way to cover your bath? I'm not sure if leaving the bath open to the air may cause some issues. 3 days may not be long enough for a whole brain. I think in the manuscript, the authors recommend about a week to clear a whole brain.

Fabio

Leaving the bath open doesnt cahnge color (for me). You have evaporation and loss of buffer volume, which has an effect on ion concentration and SDS micelles.

The problems 1 & 2 & 3, I would exclude them. The washed lipids are in the SDS micelles and are not filtered by a 5 micron filter, also you will not saturate 9L of buffer with one mouse brain.

I would run the ETC longer than 3 days for me 5-6 d at 30V, 37°C give gut results, even use 20 V.

The tissue is not clear after ETC, only after refractive index matching. My tissue is more light permissive an yellowish after ETC.

Cane the color of your buffer be because of corrosion. (parts in the circulator)?

My solution dont change color after ETC clearing.
What pH does your solution has after 3d?

XWong

Hi @Fabio, my ETC set up looks very much like yours but I'm not sure what is going on in your waterbath. Where is the pump? Is it the fish/turtle aquarium pump?
Instead of filling the waterbath with clearing solution, mine is contained in a 2L glass aspirator bottle placed inside the waterbath, and it is connected to the filter and then my electrophoresis chamber. Circulation is maintained by a peristaltic pump.

Hyewon

@liz_kritzer
liz,
We're using fish tank pump which is supposed to push about 10L of water in 1 minute.
This information is from its instruction manual, and I hadn't check the actual flow rate in this setup.
But I checked that flow rate fairly decreases with the height of chamber, so I guess flow rate is far less than 10L/min...
And um, although we tried to cover the water bath, foam was consistently generated and solution was about to overflow... but I agree with your concern..and I'll try to fix it..
Thanks for your advice!

Hyewon

@Fabio
I'm afraid there was no corrosion along the setup.
One thing I remember is that the anode platinum wire turn black after one round of circulation.
This time I'll check the pump.
And I'll also check the pH of solution.
Thanks for your advice!

By the way, your solution color didn't changed ? Oh... Can you explain more about your setup? or did you find any significant differences between your and our setup?

Hyewon

@XWong
We're using fish tank pump and it's on the bottom of our waterbath.
Oh your setup seems to be a good idea!
But is 2L enough for tissue clearing?
Last time we used 3L of clearing solution to clear up tissue but the solution got more yellowish than the picture I uploaded and brain tissue finally got swollen and turned yellow...
(Of course there were several issues with evaporation because the clearing solution was in the beaker.. Most of the solution was evaporated in the hood and by the generated foam.. )

XWong

Yes, I think I understand the way you've set up your fish pump in that waterbath. I guess you're simply using the motor head and propeller, and not the container that came with it.

As for me, I use about 1.7L of clearing solution for my ETC:
Approx. 1L inside the filter
+ 0.50L in the 2L aspirator bottle
+ 0.20L inside the electrophoresis chamber and tubings.

I filled the 2L aspirator bottle with 500mL of clearing solution only and the rest of the space keeps the foam in until it dissipates. I noticed a little bit of evaporation (maybe 100mL?), but it did not affect anything, I think.
I have over 50 hours of continuous run (20V, current fluctuating between 0.48-0.50A, 37-39C) with this set-up, and didnt notice any significant change in the color of the clearing solution and pH is at 8.5.
My mouse brain expanded after electrophoresis and a bit more in PBS-Triton-X, but it shrank back once it was placed in the little volume of FocusClear that we could afford. The transparency is comparable to the ones that other ppl show on this forum but not quite there yet.

LauraLynch

About buffer color change: I've noticed that ours turns yellowish after a few days of clearing. I change the buffer every two days (when the pH drops below 8.2).

From the looks of your photos, I think that you'll need more time for clearing with your setup, a few more days at least.

Kristin_Engberg

I agree with @Fabio that lipid saturation is very unlikely to be your problem. You may just need to run your sample for a longer time... every set up is a little bit different, so you'll likely have to calibrate how many days it takes to clear your sample for your specific setup, temperature, and voltage settings.

The tissue won't be entirely clear after ETC, but you should be able to hold it up to the light and see some transparency. In the early stages of clearing, when you hold the sample up to the light, you should be able to see a difference between the edges of the sample, where clearing has begun, and the center of the sample, which will still look dark. Do you see this with your sample?

We sometimes see a color change (yellowing) of our clearing solution after running it for several days. This may come from the inside of the circulator, or in your case, your pump. We have not noticed it to affect clearing of the tissue.

One unrelated note... it is best to have the clearing solution flow from your water bath, through the buffer filter, and then into the bottom of the ETC chamber and out the top. I see that you have it going into the top and out the bottom. The preferred flow direction is just to help prevent bubble accumulation in your sample.

Hyewon

@Kristin_Engberg
I understood the flow direction and in our setting the flow goes from waterbath - chamber - and then to filter. I think I have to change the order as you mentioned. Thanks for all your advise!

mct

@XWong

My ETC idea set up looks very much like you propose but I'm not sure where where you have to place the pump. Is it possible that you upload a image about your configuration?

Thank you in advance,