Sections for LCM-RNA

From:"Wirtala, Kelly" <>


We have been using the LCM in our lab for 2 years now.  Initially, we could
not keep the sections from washing off the slide or folding.  The most
important step in the staining is to get all of the H20 completely out of
the section. We use fresh solutions to make sure there isn't any carry over
of H20. Then we make sure the slides get ample time in 100 % ETOH.  Here is
our H&E set up:

H&E staining procedure for frozen sections (LCM)

1.	Hematoxylin, 1min (filtered) We use Mayers Hematoxylin.
2.	Tap water rinse
3.	Bluing reagent, 30 seconds
4.	70% EtOH rinse, (fresh)
5.	95% EtOH wash, (fresh)
6.	Eosin Y, 8 dips
7.	95% EtOH wash, 15 dips x 2, (fresh)
8.	100% EtOH wash, 15 dips x 3, (fresh)
9.	100% EtOH dehydration, 2 x 5min, (fresh)
10.	Xylene clearing, 3 x 5 min, (fresh)
11.	Transfer slides to blue rack and allow xylene to evaporate for at
least 5 minutes before beginning LCM

            When doing RNAse studies, all of our sloutions that require H20,
are made with Dep c H20. ( i.e Bluing, 70% ETOH, 95% ETOH).

In order to make sure my section doesn't roll when cutting, I make sure
there is ample OCT at the top and bottom of the section.
I stop my cutting stroke right before I get to the top of the section, then
bring the fly wheel back down. This leaves the section still 
attached to the mold. I use a camel hair brush to hold the bottom end of the
section and then adhere the section to my slide. 
This also allows for easy placement in the middle of the slide.

Hope this helps.

Kelly Wirtala
Fred Hutchinson Cancer Research Center

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