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).