8/21/97

Just a quick question:



I wanted to find out if anyone could tell me if they use LR White as an

embedding resin NOT for immuno work. The reason why I say not for

immuno work, is that I would like to osmicate them. I was curious to see

if I fix samples in Gluteraldehyde, and then osmicate them, could I embed

them up in LR white with Gelatin capsules/coverslips and then section

and stain for the TEM. How does this resin hold up under the beam? I

haven't seen any one mention this on this listserver, and wondered if

people even do this. Generally, I use Spurrs resin, but have some extra

LR White resin that I would like to use up before it expires.



Susan Carbyn

Atlantic Food and Horticulture Research Centre

Agriculture and Agri-Food Canada

Kentville, Nova Scotia B4N 1J5

Canada



E-mail: carbyns@em.agr.ca



Phone: (902) 679-5566

Fax: (902) 679-2311

Not as well as epoxy sections do, on uncoated grids. They can drift, tear or

flap in the electron "breeze". Solution: 1. use Formvar or similarly coated

grids to stabilize the sections. 2. Coat LR White sections (mounted on bare

grids) with thin carbon layer in a vacuum evaporator, taking care to minimize

heat delivered to sections. 3. Use higher mesh grids, eg. 200-400#.

Gib Ahlstrand, MMS Newsletter Editor

Electron Optical Facility, University of Minnesota, Dept. Plant Pathology

495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249

612-625-9728 FAX, giba@puccini.crl.umn.edu

Hi,



LR White is an acrylic embedding medium. It has many advantages for

immuno work, among them its low crosslinkage. It does not bind with the

tissue (like epoxies) but through tissue. It does not preserve tissue as

well as epoxy. It is not as beam stable as epoxy. Its polymerization

reaction is exothermic - if uncontrolled - it may damage tissue. Thick

sections may wrinkle badly (due to the lack of crosslinkage). Simply to

use LR White because it is in the refrigerator is not a good idea. For

non-immuno work it is far more advatageous to use epoxy monomers.

Bye,

hcrowley@du.edu

LR White is a good embedding medium for staright morphology after

osmication. Doesn't always section as nicely as an epoxy, but still very

useful for hard to embed materials.

G.W. Erdos, Ph.D. Phone: 352-392-1295

Scientific Director,

ICBR Electron Microscopy Core Lab

PO Box 118525 Fax: 352-846-0251

University of Florida E-mail: gwe@biotech.ufl.edu

Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/

You can use the LR White as long as you cure the resin in the oven.

The Osmium will not cause any problems. The resin holds up fine in the

beam. Just remember to exclude exposure to oxygen or the resin will not

cure.



Greg Rudomen

Greg@umic.sunysb.edu

University Microscopy Imaging Center

SUNY Stony Brook

I had some misfortunes with osmium-fixed tissue

embedded in LR White resin. The resin

polymerized prematurely during infiltration, but it

worked well with glut-fixed tissues. Someone

thought that I had too much an accelerator. The

real problem was that LR White was too old and it

reacted with osmium. A new bottle of resin worked

well for a while and then it acted up again.



I think the way to counter this problem is to buy LR

White without the accelerator already mixed.

When needed, one can mix them up and divide into

several portions for storage. A portion will be

warmed up each time and it can be used up

quickly. The rest of the resin stays cold, therefore,

it can be kept for a long time without causing

problems.



Ann Fook Yang,

EM Unit,

ECORC, Agriculture and Agri-Food Canada,

Ottawa, Ontario, Canada

K1A 0C6

YANGA@em.agr.ca

I, too, had an incident where my osmium-fixed tissue caused the LR

White to polymerize during an infiltration step. I was also infiltrating

samples of the same tissue without osmium and had no early polymerization

problems. We have not had any other problems with LR White aside from the

occasional oxygen inhibiting polymerization a bit, but we don't usually

osmicate samples for LR White embedding.

I did not add accelerator at any point during the infiltration and all

of the samples were at the same temperature.

I have not had the time to follow-up on the issue, yet.



Gregg Sobocinski

Parke-Davis Pharmaceutical Research Division

Ann Arbor, Michigan, USA

Sobocig@aa.wl.com

Just to add my own $0.02 worth here, I have also experienced this

problem. At that time I was infiltrating plant tissue, so it needed a

long time to infiltrate because of cell walls, but the LR White seemed

very non-viscous (what IS the word for non-viscous, I'm always looking

for that word), very much like water, and then, with no accelerator it

suddenly polymerized in the vials and surprized me and caused no end of

grief because of that surprize. I thought that it would slowly increase

in viscosity, such that I would be able to predict when it was going to

polymerize, and this is what fooled me. It behaved quite differently

from other resins that I've used in the past.



This was at least 6 years ago, so I cannot remember now if I had

osmicated that tissue or not.



Garry

GBurgess@exchange.hsc.mb.ca

I have run into a couple of occassions of premature polumerization

in the 10 or more years we have used LR White resin. In both cases

the resin was old, ie over 1 year but was not associated with

osmication. An possible explanation was suggested to me by Roy

Gillett of London Resin a number of years ago who definitely

recomended a shelf life of 12 months for catalysed resin.



Quote

"The reason this pre-polymerisation occurs only with tissue must be

something to do with a tissu constituent catalysing polymerisation.

Older resin is much more susceptibe to this that fresh monomer becaue

of the significant polymer growth that will inevitably have occurred

in the monomer. The most likely 'endogenousd catalyst' from

previous experience is likely to be an amine or peroxide moiety in

the tissue"



We have had no problems since switching to buying uncatalysed resin

and making up a new bottle as we run out of the old.



One point I have noticed in the discussions to date is some

ambiguity between calalyst and accelerator. From my understanding

the catalyst (benzoyl peroxide powder) must be added 24 hours before

you start using a batch of resin and is necessary for both thermal

(oven) and "cold" polymerization. The accelerator on the other hand

is added to the final resin change for rapid "cold" polymerization

without using an oven.



Ian







Ian Hallett

HortResearch

Mt Albert Research Centre

Private Bag 92 169

Auckland, New Zealand

Fax 64-9-815 4201

Telephone 64-9-849 3660

EMail ihallett@hort.cri.nz

n the case of LR White resin there is a difference in that the

catalyst powder must be added to the resin well before it is used

(at least 24h) and is used to "activate" the bottle of resin to allow

polymerisation. The shelf life of the resin starts from this time.

Initially all LR White resin was sold catalysed which caused some

problems with old stock.



The accelerator is added only when a "cold" cure is requried - I'm

not sure what it is (comes as a liquid), we never use it. The

"cold" cure is a strong exothermic reaction hence the "" around

cold.



Ian



Ian Hallett

HortResearch

Mt Albert Research Centre

Private Bag 92 169

Auckland, New Zealand

Fax 64-9-815 4201

Telephone 64-9-849 3660

EMail ihallett@hort.cri.nz

To the people discussing LR White problems



Some people mentioned that a premature polymerisation of the resin

might be avoided if the catalyst is added to the resin just prior to

the embedding.



I looked into several catalogues from different vendors but only

found kits where the resin (presumably containing the catalyst) and

the accelerator were offered.



I would like to ask for inputs which company sells LR White with the

catalyst seperate (preferably in Europe).



Hans-Martin

**************************************************************

Hans-Martin Vaihinger

Ruhr-University of Bochum

Comparative Endocrinology Research Section

Building ND 5/37

44780 Bochum

GERMANY

*********************************************************

phone ++49 234 700 4329

fax ++49 234 709 4551

e-mail Hans-Martin.Vaihinger@Ruhr-Uni-Bochum.de



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