CLARITY Cost Optimization

ecahill

Hi Everyone,

As an undergraduate engineering senior design project, we have been looking at potential for optimization of the CLARITY protocol, in terms of both time and cost. I thought I would try to get input here on what areas you would like to see explored as we spend the year working on this project. Are there particular areas that take far too long or that cost too much? We have been hearing from others that the FocusClear solution, the platinum electrodes, and the power supply are areas that we can work on. Any suggestions you have would be greatly appreciated. Of course we'll share anything useful that we find, so think of this as an opportunity to get us to explore these things for you.

Thanks!

nicolasrenier

The 3 major costs for running the protocol are (considering the hardware is available) :

- the SDS
- the platinum
- The focusclear

The focusclear costs can be reduced to practically zero by using the homemade focusclear, or a ScaleU2 solution instead. The SDS and platinum costs can be reduced to zero as well by doing passive clearing, at the expense of added time.

Then you have imaging costs, but this is a case by case thing.

Hope this helps.

KatherineHolzem

Hey all. I second the post by @nicolasrenier regarding costs. SDS is like powdered gold, and platinum... well, is platinum. I would also add that Kwik-Sil, for the imaging chambers, is surprisingly expensive too.

I have been using a few alternatives, that, thus far, have not been creating any noticeable issues:

1) SDS: I have found that prices for this vary greatly, with Sigma-Aldrich being one of the most expensive. The cheapest version I found was from Millipore (cat# 428015-1KG), at $89.00/kg.
2) Pt Wire: I have had greater luck switching to Pt-Ir (90-10) than with going with a thinner gauge wire. Currently, we are using Alfa Aesar cat# 10056. Seems to work just fine and has the added benefit of being a bit stronger than pure Pt.
3) Kwik-Sil: A quick curing elastomer is really all that's necessary, so we are trying Loctite Hysol from McMaster-Carr (cat# 7369A14). We had actually bought it to try for the ECT chamber, but it didn't withstand the detergent, but seems to be okay for the imaging chamber.

I do appreciate the FauxusClear recipes, which works great.

ecahill

With regard to the Platinum wire, does anyone have any figures available for longevity of different diameters of wire? When things go wrong with the wire, what exactly demonstrates that it's going wrong? Thinning, blackening...? Any specifics on why Pt-Ir seems to work better? Is it just ultimately producing a clearer product, or are there other factors earlier on that show it to be working better?
Thanks!

KatherineHolzem

For anyone looking for a Kwik-Sil alternative, I have found something that seems to be just as good (or maybe better) for much, much cheaper. The product is called Ecoflex 5, and it can be purchased through this website: http://www.smooth-on.com/. It's made for casting masks (for movies, Halloween, an such), but all the necessary specs (silicone rubber without shrinkage, RTV, pot life 2.5 min and demold time 12 min, flexible).

Only about $25 for 400 mL vs. about $85 for 5 mL Kwik-Sil. Scientific vendors really rip us off.

Amuoeba

Hello everyone, i was wondering if it would be possible to use external electric field to speed up lipid separation. By external i have in mind a field like in a capacitor. You would put your chamber between two metal plates connected to a high voltage DC output and an electric field would be generated between them. In this way you would no longer need expensive platinum electrodes, but i guess the power consumption would go up. If this is not posible i would be most happy to know why, because it seems that it could work, but i might be missing some parts of knowledge concerning this matter.

Kristin_Engberg

From our experience, ETC works best when the electrodes are as close to the tissue sample as possible. This forces the SDS micelles to travel through the tissue sample. When the electrodes are further apart, the micelles are more likely to travel in the excess solution, or around the sample, which is ineffective for clearing the tissue.

Clarifier

After optimizing costs based on forum advice and using what we had, here's my list of supplies that comes out to roughly ~$1,600....much better than the $11,000 original price tag

https://docs.google.com/document/d/1Enc9tadG0yhHkL_sqw9fOxY5bxbAhEk2ieXHBX0CbaM/pub

Clarifier

@KatherineHolzem Has the Pt-Ir wire given you any issues? Would you still recommend switching to it?

andas

Have anyone tried this new SDS product (74255-250G) form Sigma? It is less than a third of anything else they sell. However, it comes in pellet from (great when you weigh it), and I fear that it will take even longer to dissolve than powdered SDS. However warming up the water to about 50 °C I have dissolved 80 g/l of powdered SDS (adding it in portions).

SLJ

Has anyone tried CLARITY without ETC steps? they made ETC as optional in this papaer: "Advanced CLARITY for rapid and high-resolution imaging of intact tissues" published on June 2014.

JAGS

Hi SLJ, I'm going to begin with the passive clearing next week. If you want to discuss the experience, write me.

rchurt

I gave up on ETC a while ago. Passive clearing is great if you've got 2 weeks to wait for your samples to clear (in the case of whole mouse brains). I'll put them in 50 mL tubes of clearing solution in an incubator/shaker at ~60C and 100 RPM. I wouldn't go higher than that on the temp. Change the solution about every 2-3 days, and you should be good.

DUJ

I also gave up on ETC. The benefits of a few days faster clearing do not outweigh the disadvantages of the high cost and the pumping system you have to build. Also, we saw that antibody staining for some proteins failed for ETC processed samples suggesting that there might be some alterations of proteins. After dropping ETC, all antibodies worked. I work with kidney tissue, however.

coweight

Hi, everyone, I want to double check with you guys: when prepare the clearing solution (8%SDS in 0.1MPBS, PH7.5), I adjust the PH of 10x PBS to 7.5, then add SDS to make final concentration 8%, use microwave to help dissolve well. The solution is very viscous, and SDS will precipitate at room temperature again. Was the protocol wrong? Should I use 1x PBS instead of 10X PBS? Thank you!

WFD

@coweight My last PACT run was of the same consistency and cleared just fine, just be sure to wash out all of the SDS after clearing and you won't have any problems.

coweight

@WFD you mean your 8%SDS solution is also very viscous and hard to dissolve? Do you store it at room temperature after you made the solution? Thank you~

WFD

@coweight Yes, my 8% SDS solution never fully dissolved when preparing it at RT. However, it became very viscous, but completely dissolved and transparent, at 37 degrees Celsius during the clearing step. When returning the 8% SDS solution to RT after the clearing step, the solution recrystallized (which also occurred inside of the cleared tissue). I have been making my 8% SDS as needed for each sample and do not make large quantities to store. Multiple washes with mild shaking at RT removed the remaining SDS from the tissue and was not an issue during the immunostaining and imaging steps. Don't worry if your tissue doesn't look as clear after the washes as it did in the clearing solution; once it is incubated in FocusClear or RIMS, it should be even more transparent than it was.

Clara

nicolasrenier said:

The 3 major costs for running the protocol are (considering the hardware is available) :

- the SDS
- the platinum
- The focusclear

The focusclear costs can be reduced to practically zero by using the homemade focusclear, or a ScaleU2 solution instead. The SDS and platinum costs can be reduced to zero as well by doing passive clearing, at the expense of added time.

Then you have imaging costs, but this is a case by case thing.

Hope this helps.

Can you share the recipe for homemade FocusClear?
That would be great! Thanks

Clara

coweight said:

Hi, everyone, I want to double check with you guys: when prepare the clearing solution (8%SDS in 0.1MPBS, PH7.5), I adjust the PH of 10x PBS to 7.5, then add SDS to make final concentration 8%, use microwave to help dissolve well. The solution is very viscous, and SDS will precipitate at room temperature again. Was the protocol wrong? Should I use 1x PBS instead of 10X PBS? Thank you!

It seems that I'm missing something... Isn't the clearing solution 4% SDS in Boric Acid pH 8,5? now I'm confused...

Kristin_Engberg

Hi Clara - the clearing solution in the original CLARITY protocol is exactly as you described, 4% SDS in boric acid pH 8.5. The previous comment is referring to an alteration to the clearing solution mentioned in a paper by the Gradinaru group which introduced a modified approach to CLARITY called PACT.

Irene

Hi, i've recently published an article on a new clearing agent (TDE) suitable with CLARITY that replaces Focus Clear. It is open access, the link is http://www.nature.com/srep/2015/150507/srep09808/full/srep09808.html
Please ask me if you have any questions about it, it is very easy to do and VERY CHEAP. Furthermore it is suitable with different type of microscopy.