We have been using an IHC microglia stain on some mouse brain tissue after some experiments we ran.
We followed our labs proven protocol for staining, and we dehydrate in graded ethanol and finally place in Xylene. Several people in our lab have used this antibody and followed this protocol with no issues at all.
After coverslipping using Entellan and leaving to dry for a day or so, we are coming back to find our tissue slices all dried out.
We are using generous amounts of mounting medium when we coverslip so we are confused as to how this is happening.
Any ideas?
Thanks!
Kristie,
Datasheet of Entellan precise that the mounting medium "loss on drying = 75% ".
You have to calculate how much µL you need to cover all your coverslip regarding this important loss.
You can use another hydrophobic mounting medium to avoid this problem.
Good luck,
Virginie
Hi Kristie.
Maybe you could also renew the EtOH and xylene solutions. If you use them too often, the graded EtOH gets too much water, which also ends up into your xylene. Then the xylene gets milky, due to water insolubility. But you can´t see the EtOH in your xylene, which is more volatile. This could be the reason why your slices dried out. You can try that.
Greetings!
If you coverslip a series of sections on a series of glasses, your xylene will evaporate due to the time you need to prepare all, with the consequence of drying out.
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