Hi Everyone,
For paraffin-embedded tissues, it is highly recommended to use ethanol to
fix your samples.  The use of crosslinking fixatives (formalin, etc.) will
demand a long incubation/extraction time (>6hr).  If ethanol is used, the
extraction time is much shorter (~3hrs).  For paraffin embedded samples,
remember not to bake the sections onto the slide.  Frozen sections will
always give a higher RNA/protein recovery than paraffin embedded samples.


Procedure for deparaffinization:

1. Section paraffin blocks at 5-10 microns. 5 microns is optimal for LCM,
but the thickness should be dependant on the cell (nuclei) diameter that is
being worked on.
2. Place ribbons into a water bath at 42? to smooth out and eliminate folds
and wrinkles.
3. Mount sections onto glass slides. Air dry overnight at room temperature.
4. Dip the slide containing the tissue section in the following solutions
for the specified times.

Xylene  5 minutes
Xylene  5 minutes
100%Ethanol  30 seconds
95% Ethanol  30 seconds
70% Ethanol  30 seconds
Distilled Water 15 seconds

Enclosed in this email is a simple H&E protocol that most people use when
preparing their samples for laser capture microdissection.

Materials

70%, 95%, 100% ethanol
Xylenes,
Deionized water (nuclease free)
Hematoxylin solution, Mayer's (Sigma)
Eosin Y solution (Sigma)


Paraffin-embedded Sections or Frozen Sections

If a paraffin-embedded section is to be stained, start from step 1.
If the section was frozen-embedded, start at Step 4. Coming from -70c
freezer, allow the specimen to thaw for about 30 sec. (Do not allow section
to completely thaw, this will cause the section to stick irreversibly to the
slide)
Place the sections in the following solutions:

Fresh xylenes (to depariffinize the sections) - 5 min
Fresh xylenes - 5 min
Fresh 100% ethanol - 30 sec
Fresh 95% ethanol - 30 sec
Fresh 70% ethanol - 30 sec
Deionized water - 15 sec
Mayer's Hematoxylin - ~30 sec
Deionized water - rinse (x 2) - 10 sec
70% ethanol - 15 sec
Eosin Y - ~30 sec
Fresh 95% ethanol - 15 sec
Fresh 95% ethanol - 15 sec
Fresh 100% ethanol - 30 sec
Fresh 100% ethanol - 30 sec
Fresh xylenes (to ensure dehydration of the section) - 5 min
Air-dry for approximately 2 minutes or gently use air gun to completely
remove xylenes.
The tissue is now ready for LCM.
For successful LCM, samples must be dehydrated and stay dry.


I hope this helps.
If you have future questions, please do not hesitate to email me directly.
Sincerely,
Ashi Malekafzali





Ashi Malekafzali
Technical Application Scientist

Arcturus
400 Logue Avenue
Mountain View, CA 94043-4019
tel 650-962-3020 x310


-----Original Message-----
From: Mark Elliott [mailto:MElliott@mrl.ubc.ca]
Sent: Friday, January 31, 2003 7:13 AM
To: histonet@pathology.swmed.edu
Subject: Best fixative for LCM


I have been asked by another researcher here to posat this.  What have
people doing LCM found was the best fixative for capturing cells.
Currently our material is formalin-fixed and paraffin-embedded (using
archived tissues).  We are going to be recieving some new tissue and
will need to paraffin-embed it, however we are wondering what the best
fixative would be-formalin, Carnoy's, alcohol?- keeping in mind that we
also need good morphology.

Thanks for any and all suggestions.

W. Mark Elliott, PhD
Research Associate
UBC-McDonald  Research Lab
St. Paul's Hospital
Vancouver, BC
Canada