Re: about laser microdissection on paraffin-embeded sections

From:Todd Sherman

Hello Sean,

Don't mind at all.  Yes, the baking is to kill the RNases.  Some people
will use slides straight from the package, and for most things, that
would be fine.  However, we never assumed the sterility of any
manufacturer and decided it would be prudent to eliminate that unknown.
  The exact temperature and duration varies from lab to lab and isn't
too critical.  What I did was wrap the metal slide rack (staining rack
of 20 slides) in aluminum foil (twice because the corners tended to
puncture the foil quite readily) and bake overnight at a temperature
between 275-325F.  Our utility oven was somewhat dated and had
considerable variability.  Average of 300F seemed adequate for us and a
minimum of 3 hrs. was typical.  For convenience I loaded the slides in
the late afternoon and let them bake overnight; then, the next morning I
removed them and let them cool slowly at RT for use that afternoon.

Differentiation was about as standard as you can get.  H&E with various
hematoxylins.  The resolution with the LCM scope can be very difficult;
sadly for us, it was the norm.  If you are selecting very low incidence
RNA where only a few cells potentially contain the desired RNA, an
immuno to label desired cells might be appropriate although we never did
that.  If your cells are cancerous, as few as seven zaps (if I recall
correctly) will provide adequate material to perfrom RT-PCR.  We had to
"zap" more often and acquire more of the desired RNA that way rather
than isolating thick cells - the thinner-sectioned plane aided
visualization under the LCM since lighting can vary across the slide.
You never really know where the best region of cells is going to lie on
the slide.  Sometimes it's in the middle and sometimes its on the edge,
hence lighting and tissue adherence can vary dramatically.  Also, our
tissue under study was normal, ie. not tumor with artificially elevated
RNA levels.  Our most difficult acquisition was alveolar endothelial
cells... very difficult to visualize and isolate.  20um sections are
pretty thick and the LCM may not be able to melt off that much tissue in
one shot.  We worked with lung (5um) and getting the membrane of the cap
to set properly and fuse was tedious and sometimes problematic.  The
Arcturus model (one of the first produced since I did this about five
years ago) had a design problem in this regard in my opinion - either
that or it was not maintained properly in the host lab.  We shared the
resource and maintenance was sometimes an issue.

Another reason we avoided the immuno was that it introduced more
opportunity for contamination and it was hard on the tissue.  Some
tissue types are not very responsive to the numerous washes required to
stain them, especially when the final process is binding to an LCM
membrane.  The literature reports success performing IHC prior to LCM,
but, from what I recall, the early studies were done on cancer cells
where RNA was ubiquitous.  If only a few desired cells remained after
all of the processing, and the cells were cancerous, then that might
explain the reported successes.

Hope this helps,

Sean Trombley wrote:
> Hi Todd,
> I hope you don't mind me asking a couple of questions as well!  I am also
> interested in Harvesting RNA from FFPE tissue.  We have gotten OK results with
> our 20um sections of various tissue/species type and would like to move on to
> the LCM.  You had said that slides should be baked in an oven?  Is this to
> "kill" the Rnases?  If so how long and at what temp?  I also was wondering if
> you would be willing to share any protocols for immuno's or special stains that
> we might use to differentiate tissue types.  Thanks in advance for your help!!
> Sean
> (203)812-5471
> Bayer Corp
> Molecular and In-Vivo Pharmacology

>   Todd Sherman                                                                         
>   com>                     cc:                                                         
>                            Subject:  Re: about laser microdissection on                
>   06/18/2003 03:31          paraffin-embeded sections                                  
>   AM                                                                                   

> Hello Yichao,
> Ideally, all of your solutions used in the production of your slides
> should be "RNase free".  This means that you would probably want to use
> millipore, DEPC-treated, autoclaved water when making solutions.
> Further, and this is an unusual but desirable condition, you would
> separate your regular specimens from your research (LCM) specimens.
> This would optimally require a second tissue processor that is cleaned
> and "sterilized", ie. RNase-free.  The solutions should be as fresh as
> possible and only your work load can determine how often to rotate them.
> Next, all glassware and containers during the production of your of your
> slides must be RNase free.  Glassware, including slides, should be oven
> baked and handled with gloves at all times.  After mounting, always keep
> the slides well protected. RNAZap
> (
> comes in handy when the containers cannot be baked.  Cut and mount your
> sections the day before you are going to use the LCM.  The same day
> would be ideal but that may be asking too much from the techs.
> Typically, we mounted and stained (H&E) in the afternoon and performed
> LCM the next morning.
> These are some of the suggestions I can think of off the top of my head
> to help you control the RNases.  Not comprehensive but a beginning
> checklist.
> Good luck,
> Todd
> Yichao WU, Ph.D candidate queried:
>  >Another problem I care about mostly is how would the process of making
>  >paraffin-embeded sections destroy RNA? I believe formalin fixation
>  >would protect RNA,but How about other steps? From the incubation at 60C
>  >after sticking to slides to the deparaffinization.

Todd Sherman
HistoSoft Corporation
"Biology in a new form..."
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