These days I have some problems with the frozen section of mice brain. I used the antibody to stain the slice. Observing with a confocal microscope I found that there are a lot of black holes on the surface of slices.I think the surface of slices is uneven. Any suggestion will be welcome. Thank you.
Thank you, Saroj. I always save the slices in PBS after section. And pick some slices adhesives on the slides.
I see some black hole(no any background) on the surface of slice due to the damage of slice. Every time I use NEW blade to section the mice brain. What do you mean water droplets? Because my sample don't get sufficient dehydration.
The attachment is my picture. It is not very typical case. But you do see some hole on the picture.
Thank you for your help.
Perform H&E (or diff quick) stain on a section and so you observe under bright field. Ice crystal formation during freezing will cause artifact and is very characteristic. "Thick-thin" sections (that can producing lines, parallel to the knife) can be the result of a loose screw in the knife holder or knife assembly. Make sure everything is tight and that your frozen block is well adhered to the chuck. What thickness, and temperature are you sectioning at?
Thanks and I tend to agree with Saroj since this not present prior to staining. would add that when you pick up slides from PBS, make sure that you stand the slides upright while they dry (not flat) to help liquid drain from under section. are you doing any kind of antigen retrieval? that can also create artifact on thick sections .
Perhaps doing the procedure on free floating sections (better for thick section material) + would minimize any technical artifact that is being generated because you are transferring to slide. Also unless absolutely necessary to use thick section, try thinner frozen sections on the slide
I know such holes as "air holes" resulting from air that remains between tissue and slide during sectioning. As it does not have contact to the slide it will get lost during incubation leaving those dark spots.
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