Bio Rad Wet Transfer system for ETC?

pico

Hi Everyone,

I'm in the process of getting everything together to start (attempt) the CLARITY protocol. I was wondering if anyone had tried using a wet transfer system designed for PAGE transfer for the ETC part of the protocol (A BioRad Mini-PROTEAN Tetra System). I planned on putting my tissue in plastic histology cassettes that are normally used for tissue processing and then trapping those in the electric field with the blot holder that slides into place. I would run tubes into and out of the chamber underneath the lid where there are already holes to prevent overfilling the chamber to allow for buffer circulation.

Is this a stupid idea for a reason I can't see at the moment?

pico

Does the lack of responses indicate I'm so crazy as to not even warrant an explanation?

LinusMG

For me the lack of response means that I'm not sure :S
I think that there could be not enough flux in the chamber. For me it has been crucial for good clearing to have a high flux through the sample (~15 L/min). If you have slow flux in the chamber you can also have over-heating or bubble accumulation issues. I think that trying higher flux can generate leakage problems.

Anyway I think it's worth a try. Maybe you can compensate low flux using lower voltage (in order to avoid burning the samples) and mid-high temperature (~45ºC, in order to have better diffusion into the hydrogel). And also it would be interesting to have an "in-chamber" temperature control.

jdscott

This is exactly how I've been doing it! It works fine for me. I actually don't even circulate the buffer; I just change it out every few days. I just keep the whole thing in a water bath set to 45C, and that seems to do the trick. If you have a way to successfully circulate the buffer, I would appreciate your suggestions - I've tried a couple methods and ran into problems so it was just easier to change the buffer out. The main issue you may find is getting high enough voltage. To get higher voltage, you may try using some kind of sealant (I used a silicon glue) to seal off all exposed wires except those closest to your sample. I've gotten up to 30V this way, but I usually run it around 15V (because my power source can't handle running above about 20V for very long - personal problems!)

As to the burning issue, the tissue I'm using (liver) is robust enough that I have never had a problem with this. Everyone else I've talked to has been using a more sensitive tissue and fluid circulation or lower temperature was more critical, so you may need to make adjustments for your particular samples.

pico

Hi everyone,

Just wanted to report about how things have gone thus far. I tried out the ETC step using the biorad Xfer system and it worked great for our rat lung samples and only alright for spinal cord/brain samples.

Two reasons I can think of that the neuronal tissue isn't clearing as well...
We have a wimpy powerpack and are only able to get 15V .5A running in the chamber, less if you use a larger Xfer system. We're also not circulating the buffer since it's been hard to find a pump that will work and we don't feel like ordering one just yet. Since I really only care about the lung (the neuronal tissue was for another lab just to see how it would work) this works fine for me If you wanted to circulate the buffer (and have a pump cabable of doing it) a large Xfer chamber with space for running cooling lines would work perfectly. You'll just need a larger power pack to get more than 10V. If you use the smaller Xfer chamber, there are overflow holes that are large enough to run tubing into/out of. Alternatively, if you have a spare chamber, you can drill into the top. If I can track one down, I'm going to try it with a perfusion pump.

What I did was put the tissue in histology cassettes, trap the cassettes in the same fashion as I would a western blot, and put the whole apparatus in a water bath at 45C. After 3 hours the lungs were noticeably clearer. After 6 hours I turned I turned the ETC off so it wouldn't run overnight (I didn't want to turn things yellow) and just let everything sit in the warm clearing solution overnight. Thicker lung lobes are still somewhat cloudy but this is a test run so I want to see what happens after washing and incubation in mounting media.

Here's a link to some pics.
https://drive.google.com/folderview?id=0B0xalBg7EE2rNGp3eDI3ajJOcTg&usp=sharing

While not perfectly clear in some places. I still have the other two cassettes of lung in ETC just to see if they get clearer or just turn yellow.

Ultimately I think what I'm going to try out is buying a tissue matrix (http://www.zivicinstruments.com/) to slice the embedded tissue before ETC. I'll then place the slices in the cassettes and run them through ETC. This should speed up the process greatly and eliminate the thin/thick areas of lung that require different clearing times.

Questions/comments are welcome

Kristin_Engberg

The pictures of your lungs look great! Thanks for posting about an alternative ETC method and your insights to how different tissues respond. We often use a brain tissue matrix to slice the tissue immediately after polymerization when the tissue is the most robust. This speeds up the clearing process significantly - you may find it easiest to just do passive clearing and not bother with ETC.

hutchpd

First, thanks pico for the tank idea!! It's been perfect for testing out clearing on my mouse tissue (skin, lungs, mammary, liver) without having to invest in chamber construction.

Instead of running cooling lines (and dealing with pumps, etc), I've been having success stabilizing the temperate (in a BioRad wet transfer tank) by adding a stir bar and placing the stir plate + tank in our 4C fridge. The electrodes are routed through a port in the side of the fridge so the actual power supply is outside the fridge and excluded from condensation hazards.

To use the fridge method of cooling, the voltage needs to be limited to 20-25V, otherwise the tank still overheats.

ningtong

@pico

We tried your way of running ETC using the BioRad transfer unit. It worked, but there is a problem with the power supply. The bio-rad power supply shows resistance change detected or if using a regular power supply, overcurrent bulb is lit. Did you have similar problem? Did you use standard SBC buffer (0.2M boric acid of pH 8.5 and 4% SDS)? Or did you use a customized buffer?