I've been trying to figure out what happened but it seems I am the only one this has happened to?
Processing:
- transcardial perfusion with 1x PBS followed by 4% PFA
- post-fix brain in 4% PFA for ~24 hours in 4°
- move brain to 30% sucrose in 4° until it sinks (2-3 days)
So all was totally normal but I went to check on one of the brains that had been sitting in 30% sucrose for 3 days and the top part of the sucrose solution was frozen? I thawed it and the brain didn't sink at all, so I'm assuming the brain wasn't able to absorb any sucrose since it froze.
The brains were sitting in the top back of the 4° so it's possible that it's colder back there, but I'm struggling to understand how the cryoprotectant could freeze. I moved them to the front of the fridge after I thawed the frozen one and a day later the frozen brain had sunk to the bottom of the vial.
A couple of questions:
- how did a cryoprotectant freeze at 4°C?
- will the 30% sucrose solution freezing disrupt any signal for immunohistochemistry or disrupt the integrity of the tissue?
Hi Kate Tuite
* While 30% sucrose at 4°C should not normally freeze, but since you observed, here could be some of the reasons involved:
1. Presence of any sort of impurities in the solution.
2. Miscalculation during preparation of 30% sucrose.
3. Sometimes it may freeze if the solution is kept immediately in cold conditions.
* Yes, if the ice crystals are formed inside the brain due to the presence of water in it, that can tear and distort cells as 30% sucrose might not penetrate larger brains uniformly, leaving the core less protected.
It is always recommended to start with 10% sucrose in PBS and gradually increasing to 20% and 30% helps slow down dehydration and ensure better penetration.
Hope it helps.
Thanks,
Yes, 30% sucrose can freeze.
In your case the reason could be that some parts or your fridge are colder than others. Check the temperature of the different comparments and if is nacessary decrease the temperature of your fridge.
The reason why the brain doesn't sunk could be air bubbles in the ventricles or if you have left the dura on your brain, because it takes longer time to penetrate through this firm tissue.
If the cryoprotection is incomplete you have damaged tissue. Therefore I recommand to to at first a Nissl stain to get informations of the quality of your fixation and cryoprotection and then continue with other stainigs or immuno reactions. In the attchment you will find an example for a bad cryoprotection.
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