Using LR White™ for Electron Microscopy
Brought to you by the London Resin Company and SPI Supplies
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"Human Oral Epithelium, PTA stained"
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When using LR White™ embedding resin for dedicated electron microscopy,
very few changes need to be made to the regime used for epoxy resin embedding.
Every laboratory has its own individual embedding schedule but we have laid
out here a "typical" schedule for LR White as guidance for its use.
Fixation:
No change from normal fixation should be made, if EM only is required from
the final blocks.
If however, good ultrastructure and a wide range of LM staining is required,
then we have found that the use of freshly depolymerized paraformaldehyde (3
-4%) in a phosphate buffer pH 7.2 with 2½% w/v sucrose is the best
compromise. Glutaraldehyde along and Karnovsky's glutaraldehyde-
formaldehyde mixtures may lead to patching LM staining and some stains not
working or giving "false positives" (e. g. PAS) whereas normal formalin
fixation yields unacceptable EM ultrastructure.
For the dual LM/EM role, osmium tetroxide should be avoided due to its
effect on many LM stains but 1% phosphotungstic acid (w/v) in the first
absolute ethanol step of dehydration improves electron contrast without
adversely affecting most LM stains. Osmium tetroxide may be used if the
blocks are required for dedicated electron microscopy only.
Dehydration:
A graded ethanol series is the method of choice when embedding in LR White.
Acetone acts as a radical scavenger in the resin system and therefore traces
of acetone left in the tissue at curing can interfere with this
polymerization. For this reason the use of graded acetone series and 2,2-
dimethoxypropane (which generate acetone) are best avoided. If the use of 2
, 2-dimethoxypropane is considered vital we recommend either a protracted
resin infiltration or washing the tissue with dry ethanol prior to
infiltration in order to minimize the chance of acetone contamination of the
final resin.
Infiltration:
The extreme low viscosity of LR White may be exploited by allowing the use
of short infiltration times or large specimens but not both.
A 1 mm cube of animal tissue will be adequately infiltrated in about 3 hours
if 4-6 changes of LR White at 60° C are employed during this period. An
overnight infiltration at room temperature, followed by two short changes of
resin will often be more convenient, however. The long shelf life and low
extraction rate of LR White allows specimens (perhaps reserve tissue) to be
stored safely in resin for many weeks at 4° C if required. Larger blocks do
require significantly longer infiltration times than small ones.
Polymerization:
Osmium tetroxide reacted tissues should not be "cold cured" with the
accelerator. This process is strongly exothermic and the dark color of the
tissue leads to a local heat accumulation which can cause local problems in
and around the tissue. So we want to repeat this admonition: Do not use the accelerator
with already osmium tetroxide stained tissue. Post staining with osmium
tetroxide is quite acceptable, but only after polymerization has occurred.
If the tissue is not osmium tetroxide post-fixed then curing with LR White
accelerator may be employed. As with curing blocks for light microscopy we
recommend cooling the molds during polymerization, but there is no need to
exclude oxygen from the surface of the curing block.
Thermal curing should be used for osmicated specimens and may be used for
all specimens. Here it is important to limit the contact of oxygen with the
resin while polymerization occurs. The most convenient way of achieving
this with capsule-type embedding is to use gelatin capsules, fill up to the
brim and slide the other half of the capsule on.
If flat embedding is required for cutting orientation then the surface of
the resin must be covered to prevent contact with oxygen. One convenient
method is to utilize the JB-4-type molds and chucks, useful for light
microscopy, and after polymerization the block may be sawed off the stub and
the mold reused.
Polymerization time and temperature are fundamental to the physical
characteristics of the final block, to a much higher degree than with
undercured epoxy systems.
We strongly recommend a temperature of 60° ± 2° for a period of 20-24
hours. Some ovens are not capable of controlling polymerization temperature
so closely, and if faced with over brittle blocks, this is the first parameter
to check.
LR White has extremely good powers of penetration and can penetrate and
soften some low-density polyethylene capsules. This causes them to soften
and collapse. Also polyethylene is not impermeable to oxygen and may allow
enough contact with atmospheric oxygen to give the blocks an inhibited
"tacky" surface. Both these problems may be overcome by the use of gelatin
capsules (size 00 is similar to the popular polyethylene capsules size) and
these are much cheaper and easier to seal during polymerization.
Resin may be used straight from the refrigerator and has a very low toxicity
both in monomeric and polymerized state, unlike epoxies (see Proc. Roy. Mic.
Soc. 16, Part 4, p. 265-271). The cold cure accelerator does have some
toxic risk and contact with the skin and eyes should be avoided.
For cold curing the accelerator should be used at one drop per 10 ml of
resin and this should cause the polymerization to occur between 10-20
minutes. If polymerization occurs faster than this, we recommend either
more careful measuring of the one drop of accelerator or a higher volume of
resin per drop of accelerator.
Trimming and cutting:
Trimming the block may be done with a
jeweller's saw, razor blade
or with a glass knife on the ultramicrotome as with epoxy resin blocks.
Cutting, too, may be performed in the same way as for an epoxy resin with
glass or
diamond knives. A typical cutting
speed of about 1 mm per second is suitable.
Section Staining:
All the common section stains give good results on tissue embedded in LR
White resin. Stains made up in ethanol or methanol should be avoided as
these solvents soften the resin and may remove sections from grids. As an
alternative to uranyl acetate,
1% phosphotungstic acid has
proved a good general purpose stain, both as a block stain, as already
mentioned, and as a section stain.
In the electron microscope:
An initial reduction in electron density may accompany the initial exposure
of the resin to the beam. This is thought to represent a loss of water,
imbibed from knife-boat or staining solutions. Thinning as such does not
occur and specimens have been kept stationary under a 120 KV electron beam
for 3 hours with no obvious signs of damage.
Skin Care and Safety Products: For hand protection, consideration should be
given to the use of Skin Barrier Cream
222® and Skin
Conditioner 212®.
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Thursday February 09, 2012
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