[Histonet] New to paraffin cutting - seeking advice
modz9636 <@t> gmail.com
Mon Apr 9 19:22:15 CDT 2012
Hello everyone! I have had such wonderful feedback regarding plastic
embedded specimens and now I am moving onto paraffin. The majority of the
specimens I am going to be cutting are paraffin embedded decal rabbit
femurs. I am using a Leica RM2255 and there is an option for retraction
(after each section) and blade angle. I need to practice a lot because
right now my sections are crinkly even though I have my samples on ice, but
hopefully practice makes perfect!
So I want to ask you all a few questions regarding settings and technique
and one about H&E staining. What angle should the blade be at - I have it
set at 0 for the moment. What about the retraction option - I have it set
at 15um. The sections are being cut at 4um.
When the blade has pieces of paraffin on it, do I need to clean that off?
How do I remove this paraffin because I don't want to dull the blade? If
not, does that impact the section quality and should I be moving to a
different area of the blade?
I am looking into flotation baths that are relatively inexpensive. In
particular, I am looking for a simple bath with a glass pyrex dish. Do you
have any suggestions on where to purchase one from?
Lastly, I am going back to H&E staining of plastic embedded undecalcified
bone, specifically hematoxylin, for a brief moment. When I use a clearing
solution I know that I am trying to destain a bit from the undecalcified
bone to make a lighter stain, but does this also significantly destain the
nuclei? Right now, I need to leave the sample staining in Harris's
hematoxylin for more than 4 mins to get the nuclei stained nicely, but I
need to destain because the section is too dark. I just don't want to
destain the nuclei too much, but want a lighter stain on the undecal bone.
Again, thank you all for your support and advice, it is much appreciated!
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