Why my Clarity brain sample enlarged and become yellow

eweiren2002

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!

LinusMG

Hi,

By what you say it seems it can be normal. The pictures in the original clarity paper are black&white. It happens to become yellowish (even though not very yellow), and both in clearing solution and pbst it will be swollen (gels physical properties). It should go back to "something close to normal size" after refractive index matching.
In this paper you can see better orientative pictures: (fig 3a) http://www.nature.com/articles/srep09808

This can also be of your interest: http://eneuro.org/content/2/3/ENEURO.0022-15.2015

Best!

XIAODONG.WANG0609

Hi, sometimes my sample also turns to kind of yellow color, I run the Clarity in 4C cold room 75W, buffer temperature is around 20C. Some part of the sample turn to black if I continue running it. So, I check the sample every 1 hr and run the ETC every other day.

Leo

Hi, even under the controled 75W or 1.5mA whatever that has been applied to same sample, the clearing rate will become different by electric resistance of sample, by hardness of sample by its fixation status, by conduction due to buffer composition. The high heat value can make burning the buffer or sample. If you use different volume of chamber it can make different result. I don't know why did you use 1.5mA. It cannot be reproducibility other chamber. So you may need to find optimal A or W. Because chamber design make differet resistance. I have use 1.5A not 1.5mA. It's a 1000 folds. It works very well and I'm feeling of satisfaction as clearing speed. 6hr is enough to whole mouse brain. I also use commercial chamber.

Jake

Hi, eweiren2002

Currently, I also used a commercial Clarity Chamber.

1. You mentioned you incubated brain sample with Hydrogel solution in dark room for 2 weeks.
Did the Hydrogel solution contain PFA? In my experience, prolonged incubation of tissue samples in a PFA-containing Hydrogel extended the overall time for electrophoretic tissue clearing. If you use a PFA- and Bis-free hydrogel, 24 hr incubation will be sufficient to embed hydrogel in your tissue sample as described in Lee et al. Sci Rep 2016;6:18631. With this condition, a mouse whole brain will be cleared within 8-12 hrs.

2. Did you set 1.5 mA?
In my cases, the company who manufactured a commercial ETC chamber recommends 1.2-1.5 A.

3. Enlarged tissues after clearing
Depending on clearing techniques, tissue samples will be enlarged or shrinked. This is well-known (see Richardson & Lichtman, Cell 2015;1625:246). In case of Clarity, the tissue samples will be enlarged after clearing. However, this is not caused by electrophoresis but cased by the tissue clearing solution itself. I found that tissue samples were enlarged after incubation in tissue clearing solution even without applying electric current. The size of tissue sample will return to near its original size after incubation in a mounting solution.

4. Yellowish tissue
This phenomenon was discussed in Lee et al. Sci Rep 2016;6:18631. This may due to the Maillard reaction or an effect of endogenous chromophores. In my cases, I never experiences this with mouse whole brains.

5. Transparency of tissue after ETC
When I tried to clear a mouse whole brain first, I also expected a completely transparent tissue. But finally I realized that tissue samples is not transparent immediately after ETC. They should be cloudy because of lack of refractive index (RI) matching. The tissue samples will be completely transparent only after incubation in RI matching mounting solution. Please remember that "do not overclear you samples". Prolonged ETC may cause damage and/or burning of tissue samples.

Good Luck!