Tisssue swelling and flaking

balsorjl

Hello,

I am currently performing passive tissue clearing on an entire rat brain. It has been 2 and a half weeks, and the brain has swollen quite a bit, as was to be expected. The cortex has been cleared, yet subcortical areas remain opaque. I know that I have to be patient and wait for the passive clearing to penetrate deeper areas. However the gel has started to flake off in areas, and when I take the brain out of solution it is losing its rigidity and becoming more jelly like.

Should I keep clearing in order for the subcortical areas to become cleared? Or should I stop where I am now to avoid excessive tissue damage to the cleared parts of the brain?

LinusMG

The jellyness is normal. The clarified samples are weak and must be handled very carefully.
Could the flaking be due to any false move with the sample?
I would wait until the brain is completely clarified and only be careful with the handling.
Did you use bis-acrylamide? You can play with the acrylamide concentration in the hydrogel depending on your final goal.

balsorjl

It is possible that the flaking could be due to excessive handling, as I was taking the sample out daily to take pictures of the progress.
I used the A4P0 solution outlined in the passive clarity technique by Yang et al. (this is basically a 4% acrylamide solution). I suspect that I need to: i) adjust my acrylamide/bis-acrylamide ratio in my next sample to actually include bis-acrylamide and/or
ii) be more patient since this is a rat brain and clearing will take much longer than a mouse

Also, when my gel polymerized the consistency was between the 'flan' and 'pudding' consistency described in other forums. I decided to proceed with clearing anyway, and given that I successfully cleared cortex, I assume the gel did indeed polymerize adequately. Is it possible that this was restricted to the outer cortex, and subcortical regions did not polymerize as well? I only incubated overnight in the hydrogel solution, so I suspect longer periods of incubation (2 days) are in order to fix this.

thanks for your help LinusMG

LinusMG

From what you say it seems that the hydrogel has polymerized correctly (the consistency is correct and the timing for the part that has been cleared too).
One mouse brain takes one month to be passively cleared @37ºC.
One thing you can do in order to speed up the clearing is to increase the temperature in the clearing bath.

balsorjl

I will be sure to try this out. Thank you very much for the advice, will let you know if I am successful.