tissue not sinking in 30% sucrose

[schmiani]

I use monkey brain tissue perfused with 4% paraformaldyde. The block of brain tissue is maximal 1 cubic centimeter. It usually sinks very fast in 10% sucrose and 20% sucrose (a couple of hours each), but then does not sink to the bottom in 30% sucrose. What am I doing wrong? Or is this what usually happens?

However, it starts floating below the surface after a couple of hours in the 30% sucrose. Is it ok to already use it for doing frozen sections by then? I need to do the sections as soon as possible, as the staining procedure I am aiming for (cytochrome oxidase) needs the brain to be as fresh as possible.

Thanks for any tips!

[excalibur]

How long did you fix after perfusion?

[schmiani]

I tried different times for postfixing: 2h to 24h. It didn't seem to make a difference, it just doesn't sink to the bottom in the 30% sucrose.

Could it be that using too small a container (about 5 ml solution for a block of brain of 1 cc) is the problem?

Thanks for your help!

[CanuckPhD]

I usually use a 50 ml Falcon tube for my sucrose protection, so try a larger volume of solution. As well, gently invert the tube a few times as high concentrations of sucrose (e.g 25-30%) can form gradients. This may mean that your tissue is sitting at an interface of a high and low sucrose.

In the end though the tissue may not sink all the way to the bottom. I sometimes find this when I do whole rat brains. However, the tissue does seem to cut okay and have decent morphology.

Good luck.

[schmiani]

Thanks, I will try this next time!

I just cut the tissue that didn't sink in 30% and it was perfectly fine, so maybe it just doesn't matter.

[Cardio]

The only suggestion I have if your worriered if the tissue isn't properly dehydrated is freezing it in liquid nitrogen although if you do this I would suggest asking me how as it can crack a block of tissue if done wrong. This will help morphology and prevent crystals.

The only experience I have of tissue not sinking is bubbles in side the chamber of mouse heart. Give the tissue a slight sqeeze with forceps normally helps me.