Re: LR White protocol

<< Previous Message | Next Message >>
From:"R.Wadley" <>
Date:Tue, 27 Jul 1999 10:09:21 +1000
Content-Type:text/plain; charset="us-ascii"

	Dear Rena,

	I assume you are attempting immuno EM.  LR White is a quite corrosive &
extractive resin, & at an EM level you have to trade off good
immunogenicity for very poor morphology.  If you are using biopolymers
(implant materials) you may find they are structurally altered or disolved
by this resin.

	Generally, 2-4% paraformaldehyde + 0.5-2% glutaraldehyde in an appropriate
buffer.  (I use the 4/2 combo)
	Note: too little glutaraldehyde has been reported to be as bad as too much.
	DO NOT use  OsO4.  Picric acid can be used as a substitute (I'll have to
find my reference).

	Only partial dehydration to 70% ethanol is required.

	50:50 70% ethanol/LR white, for a few hours followed by pure LR Wite
resin.  Because the ethanol & LR White are good at stripping intracellular
components try to keep these steps as short as possible.  The time invilved
depends on the size & density of your tissue.

	It is essential that O2 is excluded during the polymerisation process.
BEEM capsules are O2 permiable, so be choosy as to your method.  Gelatin
capsules are ideal.  Most cell culture plates dissolve!  Be careful if you
use vaccuum to remove O2, you can boil your resin creating lots of holes in
the block.
	Do not use excessive heat if you are doing immuno work.  
	Do not use to much accelerator if any at all.
	LR White will cure very nicely outside on a sunny window sill.

	Glass or steel (disposables too).  Glass obviously gives the best (&
cheapest) result.  Sapphire & diamond knives are excellent.  Generally your
sections should stick down to the glass slide OK.  You can either treat you
LR WHite section as a very big 'thick' & put it on a drop of water (20%
ethanol) on a slide then put it on a hot plate, or pick it up off a water
bath as per a paraffin section.

	Just like a frozen, depending on thickness, extra time might be required
in Haematoxylin.  Eosin will stain the resin, but can be washed out to give
pink cell bits & a very pale pink background.  If you take too long to
stain you might find the section takes up enough water to drop off.

	Now if your really keen, you can find the bit you want by LM, then
re-embed the section on the slide, break it off, & re-section for EM.

	Hope this helps, let me know if you want more info or some more references
(I'll list those for all when I find them).


	Rob W.

At 02:39 PM 7/26/99 -0500, you wrote:
>Does anyone out there have a protocol they use for processing tissue in
>LR White acrylic resin so it can be used for H&E stained slides as well
>as TEM sections?  I am looking for information all the way from fixation
>through tissue staining.  I have a general guide that comes with the
>resin, but I am hoping to find someone who has made the process work.
>Thanks for your help.
>Rena Krol

R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591

<< Previous Message | Next Message >>