Electrophoretic Tissue Clearing (ETC) — CLARITY forum

Issue with Fluorophore Quenching

stephen_james — Fri, 14 Jun 2019 08:52:20 +0000

Here is the link to the protocol, on which we have based our approach; however we have made a few alterations: https://docs.abcam.com/pdf/protocols/clarity-protocol.pdf The hydrogel embedding and custom-built ETC whole-brain tissue-clearing parts of the protocol have shown successful results; however, when imaging the clarified samples either with light-sheet or confocal microscopy, the fluorophore (e.g.; monosynaptic GFP-Rabies virus, transgenic mice) seems to have been completely quenched. The following parameters are used: Constant current of 1.5A which stabilizes around 55V Temperature alternating between 30˚C and 40˚C pH level beginning around 8.5 and dropping to 7.3 within two to three hours The following links show the custom built setup: Setup_FrontView: https://drive.google.com/file/d/1n0vf5S9_CYRD-SgXB2cFFb2Ifqf-Do2a/view?usp=sharing Setup_TopView: https://drive.google.com/file/d/13ktnk1liz6k54j39Cxe-_ON0x3bNhQyd/view?usp=sharing Setup_SideView: https://drive.google.com/file/d/19fXu5x7-C4ughs40r5XbKhv1kSN0JksM/view?usp=sharing **Note: The filter component has been replaced with a 60mL Nalgene container, in which the SDS solution (1L H2Omq, 200mM SDS, 20mM lithium hydroxide monohydrate, and 0.5% 1-thioglycerol) passes through a Falcon cell strainer before reaching the sample. Secondly, the reservoir has been replaced with a 1L Nalgene container. Questions: 1. I have been using a constant 1.5A and 1L of solution as suggested by (Lee, et al. 2016; DOI: 10.1038/srep18631); however, in papers such as (Kim, et al 2018; DOI:10.1038/s41598-018-31153-7) and (Kim, et al 2018; DOI:10.1038/s41598-018-26776-9) it lists the better parameter as 1.5mA. Is this a typo? 2. It has been demonstrated that using platinum plates, instead of platinum wires, leads to a slower decrease in clearing solution pH level. Does anyone know a vendor who sells platinum plates? 3. Since we have seen a continuing quenching of the fluorophore after active clarification, what are the main parameters in the active CLARITY process that could be contributing to fluorophore quenching? Should we consider adding Bis-Acrylamide into the hydrogel monomer solution to induce a stronger cross-linking? Should we consider leaving the active method, and switch to PaCT? Thank you for your time and help! I'd be happy to provide any further information.

Need some help on our ETC setup....

Hyewon — Tue, 06 Aug 2013 10:21:57 +0000

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?

How transparent the tissue will be after ETC or passive clear?

Jing — Sat, 03 Mar 2018 19:11:06 +0000

Is it still a little white or it needs to be totally transparent? How to know it's cleared enough? I don't know when to stop the clearing process. Does anyone know when to stop is better?

New clarification service - What do you think?

JLWNeuro — Tue, 24 Oct 2017 19:05:55 +0000

Hey everyone, We have developed an improved clarification technology that allows us to clarify with really high quality and in a very short time. For example, we can do a full mouse brain in a few days. We are turning this into a clarification service for research labs around the world. Our site: claritytechlab.com (in progress) As CLARITY users for many years, our team has gone through the challenges that come with setting up CLARITY in our labs. As you are all aware, the toughest part is clarification. We have invested countless hours and funds improving this step as it is the most important and time-consuming. What do you think of a one-stop service that clarifies your extracted samples at the highest quality and sends them back ready to image in under 5 days? We would love your thoughts on this and if you would be interested. Our goal is to launch in March 2018 and help labs implement CLARITY easily. Cheers, Justin L.W.

New Tissue Clearing System (COrES)

aysegul — Fri, 07 Apr 2017 08:26:51 +0000

Hi Everyone, We developed new tissue clearing system which works as a whole. This system is an all-in-one and ready-to-use to make electrophoretic tissue clearing simple, rapid, and efficient. The system design performed by our research team was formed as a whole include electronic control unit, liquid tank, temperature control unit of the tank and electrophoresis chamber. The control unit software was produced according to feedback principle to keep the system stable using information of temperature and circulation rate received from system through the sensors. In this way, circulation rate, temperature values and current given to the electrophoresis chamber can be monitored and controlled via the touchscreen. Processing operations can be monitored through the transparent areas on both sides of the electrophoresis chamber that specially designed and produced by 3D printer and cage, putting the tissue in it, can be attachable and detachable in the electrophoresis chamber. In this way, the tissue is prevented being damaged during placing and withdrawal of it. Corrosion and particle collection is reduced to a minimum because of the envisaged electrodes for electrophoresis is produced using platinum wire. Temperature control unit is a sensitive, controlled design and does not require compressors, unlike the commonly used systems it is capable of both heating and cooling process from liquid tank base. System successfully produces transparent tissues about 3-4 days cleared tissues are efficiently labeled with antibodies. I can share the details with interested people.

Clearing Pancreas Tissue Using ETC

patiprat — Fri, 13 Sep 2013 20:45:46 +0000

Hello All I am interested in knowing if anyone in this group was able to clear pancreas tissue using clarity setup, if so what were the conditions you used for the clearing step. I have been clearing two mice pancreas at 25 V 37 degrees for 72 hrs now but I see only partial clearing in my tissue. Here is the link to the pictures: https://plus.google.com/photos/102653309454259671450/albums/5923204360943835793?authkey=CI3M8quwoM-FFg I am continuing to clear the tissue after 72 hrs. I would like to know if you have had any success with clearing pancreas and if you could share insights on clearing this tissue type it would be very helpful. Thank you all for contributing to this forum thus far, I have found the threads in this forum very useful.

New Inexpensive ETC Chamber

ONEcore — Wed, 21 Sep 2016 16:39:35 +0000

The Optogenetics and Neural Engineering (ONE) Core of the University of Colorado has designed a new ETC chamber made mostly from ‘off the shelf’ plumbing parts. Compared to other chamber designs, this design is easy to fabricate, is less prone to leaking, is easily expandable, better secures the tissue, deals well with bubbles, and is inexpensive (less than $65/chamber). The clearing solution flows in the same direction as the current, and is therefore called Linear ETC. This leads to efficient clearing (half mouse brain in two days). More information can be found in the .pdf posted to dropbox below (no account necessary). https://www.dropbox.com/s/0r5ojcit6kgeh9l/Linear%20CLARITY%20Electrophoretic%20Tissue%20Clearing%20Chamber.pdf?dl=0 Please acknowledge the ONE Core facility in your publications. An appropriate wording would be: "Engineering support was provided by the Optogenetics and Neural Engineering Core at the University of Colorado Anschutz Medical Campus, funded in part by the National Institute for Neurological Disorders and Stroke of the National Institutes of Health under award number P30NS048154."

Cooked brain made me sad

cytokine — Wed, 17 Aug 2016 14:57:35 +0000

We have just tried running clearing using a peristaltic pump and had the same unfortunate outcome that others have noted - the tubing cracked o/n, leaked, brain cooked, mop recruited from corner. I saw that someone suggested Excelprene TPE tubing - has anyone tried this and found that it stands up to 600rpm (gives me around 500mL/min) without cracking? Are there alternatives (pump, tubing, method, etc). thank you.

ETC setup - circulating PUMP for clearing solution

richik — Wed, 08 Jun 2016 20:18:05 +0000

Hi, I have recently started performing CLARITY on Xenopus frog embryos. I was wondering what kind of circulation pump I could use to circulate the clearing buffer at flow rate of 1- 2 L/min? I only have a peristaltic pump which can circulate at 500ml/hr max. Any suggestion about the particular pump's name and manufacturer would be really helpful!

Would passive clearing reduce tissue expansion compared to ETC?

xyang — Thu, 05 May 2016 21:28:43 +0000

Hello, My sample is 1 mm-thick mouse brain slice, and I really want to avoid tissue expansion during the clearing process (even it finally shrink back to the original size in the RI matching solution). So I was wondering whether I should use ETC or passive clearing? Would passive clearing have less tissue expansion compared to ETC? Since my brain slice is relative thin, how long will passive clearing take? Any suggestion would be highly appreciated!

Whole brain clarity 48hrs ETC

Neon — Mon, 30 Dec 2013 08:28:04 +0000

With a modified chamber design and electric condition, I got the fast ETC result. 1A, 30V used for 48hrs. https://drive.google.com/#folders/0B5k-H33OqLCjdUduNnJ4dV9wcUE

Stochastic Electrotransport

jrbutler — Fri, 15 Apr 2016 20:31:56 +0000

Has anyone taken a look at the recent paper from the Chung lab, "Stochastic electrotransport selectively enhances the transport of highly electromobile molecules"? It offers some solutions for some of the challenges of electrophoretic clearing, and I was wondering what people thought of it? You can find the abstract here: http://www.pnas.org/content/112/46/E6274.short Thanks, Jeff

Why my Clarity brain sample enlarged and become yellow

eweiren2002 — Wed, 06 Jan 2016 05:38:33 +0000

This is my first try to do Clarity with 2M mouse whole brain. The brain sample was perfused and fixed with 4% PFA. So I incubated brain sample with Hydrogel solution in dark room for 2 weeks on a rotator. After polymerization and washing, I did electrophoretic tissue clearing with one commercial Clarity Chamber, the set is 37 degree, and 1.5mA. After 2 days, I checked the brain sample, and found the size of brain enlarged almost twice and color become yellow, kind of semi-transparents. Anyone know what caused this? specially yellow color? Thanks!

Has anybody done CLARITY in peripheral nerves?

JosephMarmerstein — Mon, 04 Jan 2016 16:28:30 +0000

We'd potentially like to try doing CLARITY in peripheral nerves. Haven't been able to find anybody that's done so on these forums. Having done some CLARITY in the past I'd like to avoid doing ETC if possible, since I know it can add some complications. Do you think small nerve samples (<200um diameter) will clear passively? If anyone has any experience with this, I'd appreciate any answers!

pH of clearing solution

mullanekate — Wed, 26 Nov 2014 15:16:11 +0000

Does anyone know the reason behind the clearing solution having to be at a pH of 8.5?

Bio Rad Wet Transfer system for ETC?

pico — Wed, 26 Feb 2014 15:12:26 +0000

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?

Other types of tissue

abachman — Mon, 24 Jun 2013 14:43:25 +0000

Has anyone had any experience trying the CLARITY protocol on tissue other than Brain or spinal cord? We were hoping we could use this approach to examine the clonality of fat pad development and/or clonality in solid tumors. I'm concerned we will build a chamber only to find we simply "cleared" away our adipose tissue... If anyone has experience with other tissue types I would love to hear how it went!

ETC currents and shock hazard

mhurry — Mon, 09 Mar 2015 14:42:47 +0000

We are just setting up the components for ETC and I have a question about safety. I noticed people are running currents of up to 800mA through the circulating clearing solution. How safe is this and what measures do you take to prevent shocks? Our water circulator is metal - will this become live during electrophoresis and become a shock hazard? Sorry if there is an obvious answer to this question, but I can't see it addressed anywhere else in the forum/literature!

SDS and acrylamide concentrations

pavlovaip — Thu, 05 Feb 2015 16:24:27 +0000

Has anyone done ETC clearing with more than 4% SDS such as 8% or 20% SDS? Did this result in faster clearing time and was it safe for the tissue? Also, has anyone tried to use 8% SDS and 1% acrylamide (in the HM solution) successfully and what was the maximum time needed to clear 1/2 of a brain?

Post pictures of cleared tissue

Suzschindler — Tue, 06 Aug 2013 17:52:55 +0000

Please post some pictures of your cleared brains, including the conditions under which you ran the ETC (20 V x 7 days).

How much Pt wire do you actually use?

hydrazine — Mon, 06 Oct 2014 20:59:32 +0000

I'm looking into buying some Pt wire to do CLARITY and am finding some seemingly good deals on amazon. The protocol says ~50 cm. Do you guys find you use that length for the electrodes or more or less? Thanks.

Clear after ETC but turbid after PBST washing

DominicJG — Tue, 18 Mar 2014 13:42:54 +0000

Hi everyone, I would like to share my current experience on clearing mouse brain. I was also wondering what are your thoughts on these points? 1. After clearing mouse brain (23V, 1.03A, 37C), I can easily look through when holding the sample against daylight. However, when I continue following the protocol with washing the sample in PBST (24hrs x 2), the tissue turns turbid (which resolves after mounting). 2. I usually mount my sample in 80% glycerol to see if the clearing was sufficient and only for imaging in focusclear. The mounted tissue usually becomes pretty translucent. However, my last sample was very clear at the first couple of hours. When I looked at it the next day, it almost seemed like it has never been cleared at all. I am wondering if anyone has similar experience? Looking forward to reading your comments. Thank you

3D printed glazed ceramic ETC clearing chamber

LabDaemons — Wed, 24 Jul 2013 04:25:21 +0000

Hey everyone. I saw a prior thread discussing a 3D printed ETC chamber and would like to jump in with my own contribution. I've also been working on a 3D printed design for a couple of months and it is finally at the point where I'm comfortable offering it for others to use. The key difference between this chamber and other 3D printed chambers is that it's made from glazed ceramic. I decided to go with this print material instead of extruded thermoplastic for a number of reasons: 1) heat tolerant; 2) chemically resistant; 3) mechanically stronger (no warping under pressure); 4) 100% water proof; 5) 100% light proof to protect photosensitive samples; 6) much better resolution and tolerances (negligible variability between prints) I've printed and mechanically tested various prototypes (lab is setting up to run the full CLARITY protocol but not quite ready yet) and can say that it is water tight under typical ETC pressures. I'd love for someone who is already up and running with the CLARITY protocol to try clearing a brain in it! The current design has some nice features and optimizations (see photos on Flickr at http://flic.kr/s/aHsjH3Q3MC): 1) chamfered internal edges to minimize trapped bubbles; 2) use of a flat rubber gasket instead of an o-ring to provide a reliable water tight lid seal; 3) parallel electrode ports to make electrode insertion easier; 4) adjustable/replaceable electrodes w/ custom gaskets to prevent leaking; 5) simple and foolproof lid securing system using two rubber bands The chamber is available on Shapeways (http://tinyurl.com/m2qjana) in multiple colors. In the spirit of open hardware you can freely download the .stl file from that site as well if you want to try printing it on your own. I can't host the original design files on Shapeways, but if you'd like access to those just shoot me a message. Full disclosure: you'd save about $7 by printing it yourself from the .stl file, but that little extra helps me design and pay for prototypes to improve this design and develop other awesome open science projects. Hope you guys like it!

Tissue discoloration into shades of green

Asia24 — Mon, 23 Jun 2014 19:50:46 +0000

Dear All, I started the ECT procedure and after few days of running a rat brain tissue block (no overnight run) we started to notice that our brain turned greenish in its color. there was definitely some progress in the clearance, but not spectacular and this discolorations progressed in time. As recommended I cleaned the electrodes by reversing the current every 2-3 days. Initially i though it may be the black deposits entering the tissue. We did see that our cathode turned back very soon after the initiation of the run. After this experience I started running a new piece of tissue and cleaning the electrodes every day before starting the system (we don't run it overnight). And again after 3 days my tissue showed this dark 'greenish' discoloration. Does anyone have any idea what this could by caused by? Could this mean that the filter is not catching all the small particles coming of the electrode during cleaning and they stick to my tissue? Some facts: - We run our system with platinum electrodes. - We run one chamber using about 5L of SDS buffer - I check the pH of buffer regularly and its around 8.5 - the buffer is clear (no color change as seen by others) and even under the microscope i see no floating particles (maybe they are too small?) I would greatly appreciate any comments. Thanks, Asia

Filter and Circulator settings

Andy_Donaldson — Tue, 18 Jun 2013 16:14:52 +0000

Hello, I have just a few quick questions regarding the buffer filter and the settings for the water circulator during the ETC step. First of all, how often do you replace the filter cartridges? I am in the process of assembling my apparatus and I'm not sure how many replacement cartridges to order. Secondly, what level do you have your pump flow/pressure at during ETC? Do you change out your clearing solution at any point during ETC? Finally, did/does your circulator have any problems with circulating the clearing solution? Thanks so much for your help.

Problems with ELECTROPHORESIS SETUP

Vortexam — Thu, 26 Jun 2014 14:29:34 +0000

I recently started setting up clarity protocol. I don't have a temperature controlled water circulator.Instead I put the clearing solution directly into the water bath which outlet is connected to inlet of the chamber of my tissue I am facing trouble in keeping voltage at 25V,my electrophoresis power supply is not from biorad and it is not having any power pac adaptor. Can anyone please help,and put a photo of there assembly,just for some guidance. Thank You

When to change SDS buffer while running ETC

Shashank20 — Mon, 20 Jan 2014 13:11:58 +0000

Hi everyone I am running ETC for clearing mouse brains. I am not sure if one can reuse the pre-used SDS buffer (7-8Litres used to clear 1 mouse brain 30V/37oC) for clearing another couple of brains or one has to use fresh buffer every time when clearing brain. Though the pH of the used buffer is fairly constant, but I am not sure how one can now if the buffer is saturated with lipids. Re-use of SDS buffer will lower down the running cost. Any suggestions are most welcome ?? thanks

Surface cracks?

Tully — Mon, 16 Jun 2014 11:35:56 +0000

I'm currently clearing an entire right hemisphere from a rat brain. I took the brain out of the ETC chamber today to check the clearing status, and I noticed quite a few cracks on the surface of the brain. I'm not really sure if its due to the motion from all the bubbles in the chamber, or the slight force needed to get it in and out of the small chamber that "holds" it within the ETC chamber, or temperature or what. I'm running the ETC at 20V, at roughly 35C (measured from within the chamber, room temp is about 25c, so the difference shouldn't be huge), with a flow of about 0.5L per minute. It has been running since june 12th (16th today), with an ETC pause over the weekend (14-15th june) running passive only. When running passive its at roughly 28c. Has anyone had any similar experience with surface cracks?

brass in contact with buffer

arvid — Thu, 01 Aug 2013 12:27:55 +0000

I have brass in contact with the buffer at one point in my system, and have noticed that it is getting darker over time. What kind of metals are in contact with the buffer in your systems? Brass? Stainless steel? Aluminium? Does anyone know how it is effected by the buffer, and how it effects ETC?

Loss of GFP Fluorescence

gjones — Fri, 25 Apr 2014 00:22:48 +0000

Hi All, With all of the brains we've been clearing, we're noticing that all of the endogenous GFP leaves the cell and is somehow instead staining everything but the cells that originally expressed the GFP. This has happened every time so far and we've tried making new solutions with brand new reagents, as well as just using passive diffusion to clear the tissue instead of the tissue electrophoresis, however each and every time we image the sample, the GFP has left the cell and is instead sitting in the surrounding tissue. We've tried this now on a GAD67 and Thy1 models with the same result. The tissue clearing itself appears to work perfectly, just not the imaging/retention of GFP. Does anyone have any idea what could be going on??