Critical details of the protocol

LinusMG

Hello everyone,

I am trying for the first time the CLARITY protocol as a part of my recently started PhD project. Since some months ago we have been gathering all the needed material and from approximately three weeks ago we started with 4 perfusions. Until today it seemed it was going good enough unless for two bubbles in the ventricles. Today we have looked for fluorescence in our first sample (in ScaleU2) but we couldn't see any signal, maybe because of a not long enough ETC clearing step.

Implementing the protocol with the first brain I have come up with two critical doubts:

- Pump flow rate: We are using a Lauda Eco RE 415. I started using a ~15L/min (At 37ºC and 25V) flow rate in order to avoid having bubbles in the inlet of the ETC chamber. After 8 days I switched to a slower flow rate (~10L/min). I noticed that using the slower flow rate clearing seemed to be more efficient.
(If anyone is using the same pump I chose the settings 3 and then 2 in the pump power menu.)

My question here is: there is any agreement about the optimal flow rate for a range of settings between 20-30V and at 37ºC? @joelrosiene suggested to use flow rates lower than 180mL/min. @nicolasrenier reports big bubbles trapped in the ventricles using the maximum flow rate allowed by Lauda (maybe caused by a not gentle start after setting the sample into the chamber?). Also @Neon reports 48hrs ETC using 20-30ml/min and 30V.
A second question is: Has anybody used successfully the maximum values suggested in the original article (50ºC and 60V)?

- Mounting media for imaging: We decided to use ScaleU2 after reading the advances by @LauraLynch using Fauxcusclear. Anyway @joelrosiene also reports loss of signal using ScaleU2 (could it be that the slower flow rate that we used has something to do with the loss of signal?) .

@LauraLynch could you confirm that you don't have loss of signal using ScaleU2?
@nicolasrenier did you have any luck with other recipes following the FocusClear patent?
@Neon did you successfully image your 48hrs cleared samples?

Thank you all for your insightful contributions in this forum.



P.S.: A link for some pictures of our setup and sample:
https://plus.google.com/photos/109959696841588744124/albums/5975050760506825313?authkey=CIGZwO717-n_OA

We have been running ETC clearing for 11 days and probably I have been too impatient washing and incubating in ScaleU2 (the brain was translucent but not very clear).

LauraLynch

@LinusMG: We haven't imaged with ScaleU2.

nicolasrenier

@LinusMG: sorry, I have not tried to further develop the recipes for focusclear. After extensive testings, I am now using 3-DISCO exclusively for my experiments, which works better than clarity for my particular applications (mostly embryo work and alexa-CTB tracings in adults).

joelrosiene

@LinusMG My first instinct is to say that flow rate is primarily relevant to the management of the temperature of the chamber. If you can get away with a lower flow rate and still hold the temperature steady, you might want to do so so as to prevent bubbles being introduced to the tissue.

That said, 50C and 60V seems quite high - last time I did that I was unable to keep the temperature down, and my PVC chamber became clouded and leaky. Also, the sample melted I am currently clearing a brain with some very nice bright signal, and I plan to mount in ScaleA2 for imaging.

LinusMG

Hello again,

Thank you all for your answers.

After some trials I have seen that the flow rate I used was too low, even at low voltages I was burning the samples before enough clearing (I think you are right @joelrosiene). Now I use the maximum flow rate allowed by the Lauda pump (~20L/min). Gradually starting and stopping the flux I have not had any bubbles in the samples.

I also decided to try the maximum values I could (I was not able to use voltages higher than 40V (2A)). One of the best cleared samples I had, has been passively clearing for one month an after 24 hours of ETC at 40V and 50ºC.
Using the same values for freshly embedded samples, they were burnt after few days.

Now I started using 30V (amperage oscillates between 0.9A and 1.5A) and 50ºC for 2 days and after that some days (4-5) of passive clearing (Kristin_Engberg post in "Incubation time in SDS clearing solution" discussion). It seems I have the best results in this last case.

About the index matching I have already explained it in the "FocusClear alternative" discussion:
1.- I got rid of the precipitates forming into the samples (when using glycerol) incubating for some time at 45ºC.
2.- I used ScaleU2 again and there is no loss of fluorescence signal. Anyway the results are similar than in glycerol.
3.- I tried using 50%-glycerol, 80% and ScaleU2 I post three pictures of the same sample (sample 2 in the link) in a stereomicroscope (2days in ETC 30V 50ºC, 4 passively clearing after ETC). Maybe I will find more substantial differences when scanning deep into the samples.
(@LauraLynch it would be great to compare if you have any pictures like these from your samples)

Finally I have some issues with black carbonaceous residues on the side of the sample that faces the cathode. I started to improve the isolation of the samples in the chamber. By now I am using the bottom of a second sample holder for enclosing them and it seems it works. I have tried to physically clean the electrodes when I change the samples. I will also start to use the change of polarity that Kristin_Engberg explains in "Questions on CLARITY process" discussion.

We obtained an OPT reconstruction with the sample 1 shown in the link (1month passively clearing + 1 day ETC 40V[2A] 50ºC) but it is kind of useless because of the autofluorescence of the sample and the resolution of the technique. Now that I start to have good clearing we are working in order to try SPIM and confocal imaging.


https://drive.google.com/folderview?id=0B9SnslqET-pLRWJiNzVzWm1jeTQ&usp=sharing

LinusMG

Hi again,

After some months I can say that this finally works fine. Using the parameters I said in the last comment and mounting the samples in 87% glycerol + DABCO (2.5 mg/ml) (as suggested by @raju in "Autofluorescence" discussion), we have been able to nicely image two samples using SPIM. I post a link to a video and a picture of the nicest dataset (both low resolution) showing part of the hippocampus and cortex. The stack shown is the result of stitching 8 single stacks, the depth of the stacks is roughly 4mm (the image starts to be blurry approximately at this point). The picture is a depth-cued substack of ~400um.

https://drive.google.com/folderview?id=0B9SnslqET-pLcXcyX3JLc1dGSms&usp=sharing

I suppose that a lot of people has already arrived to this point, but I wanted to make clear that glycerol is a suitable alternative to Focusclear.

Fabio

Hi,

congrats your video looks pretty good. I am planing also in using a SPIM for imaging. What kind of SPIM did you use? a self.made or a commercial one like the Lavision one?

LinusMG

Hi Fabio,

It is self-made (the imaging has been in collaboration with Jim Swoger, in James Sharpe's lab). If you are interested in further details about the SPIM set-up just write me and I'll try my best to help.
Also, the Lavision you mention will solve your problem if you are not interested in features below their resolution limit, ~5um I think.

Genesprite

Beautiful Linus!

mct

Hi @LinusMG ,

I like you some years ago, I am trying for the first time the CLARITY protocol as part of my Phd project. In the first place, thanks for your ideas.

I tried to see you images (drive) but I can´t do it. Do you uploadthis images (configuration ECT system) again?

Thank you in advance,