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Author Topic: Lizard hatchling brains falling off slides during IHC staining  (Read 1910 times)

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Offline EDavid

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Hey everyone,

I have been running into an issue trying to stain lizard hatchling brains. The brains were fixed in PFA and then sunk in sucrose in a fridge. I am slicing at 50um in a cryostat and I mount the slices to the slides. I have tried gel coated and Superfrost ++ slides and gel coated is working better, but I am still losing over 50% of the slices during the staining process. I have tried letting them dry at 36C in an incubator and at 40C on a hot plate for 24 and 36 hours, and the hotplate is a little better then the incubator. I have also tried mounting the brain to the chuck with PBS and OCT, but have not found much effect of that (PBS might stay a little better, but with OCT we get much better slices).

I have also done adult lizard brains, before and did not have this issue, so I am not sure what I could try to do differently to get the slices to stay on the slide. I am doing the mounted staining because the slices are so small (diameter <0.4mm) and delicate that trying to float stain them either ends up with us loosing the slice or it becoming to damaged to use. Any ideas on what to try to get the slices to stay on the slide would be wonderful, as I am running out of ideas.