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?
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.
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
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.