LR White Embedding Kit (Medium and Hard)

C025 & C026

LR White Resin (1981 is an aromatic acrylic resin mixture which was introduced  following the work by Causton, Gillett and Germain, 1980)– It is a very low viscosity (8 cps), low toxicity, beam stable resin. It is a hydrophilic embedding medium and the sections of polymerised LR White resin are hydrophilic. This character allows immuno cytochemistry reagents to easily penetrate into the section without the need of etching, (etching sections can effect delicate tissue antigens).

LR White can be polymerised by four different methods:

1. Heat: 60 – 65°C
2. UV irradiation (365 nm wavelength)
3. Chemical with accelerator
4. Microwave

EMS supplies LR White in either the premixed form - ready to use (Catalyzed LR White), which makes it convenient for the end user or the non-catalyzed version with 9.9g of Catalyst which accompanies the LR White resin with instructions showing you how to catalyze the resin prior to use. (Usually it takes 12 hours at room temperature and 12 hours in the refrigerator for catalyzed LR White resin to take effect prior to use)

LR White (catalysed) should be kept at 4°C or lower. In our experience, the shelf-life of LR White is a minimum of one year @ 4°C, and up to 15 months if stored as low as -80°C. If the resin is frozen, allow it to thaw and reach  room temperature prior to use. If the viscosity of the LR White resin changes (becomes thicker), stop and do not use it.

Using LR White for Electron Microscopy Protocols

Fixation

Conventional EM/LM: 2 to 4 hours - recommended Paraformaldehyde (3 – 4%) solution in 0.1M sodium phosphate buffer, pH 7.2. Avoid using  Glutaraldehyde alone or Karnovsky's glutaraldehyde/formaldehyde mixture, for this may lead to a patchy stain  or some stains not working well.

For Microwave: Place specimen cubes (~1 to 1.5 mm2) in 1.5ml centrifuge tube with ~600 µl 4% Paraformadehyde in Sodium Phosphate buffer pH 7.2. Microwave irradiate  the vial for 40 seconds in a cold spot. Let the vial + tissue + buffer sit for an additional  5 minutes allowing for the cooling of  the buffer ≤20°C, and then irradiate again the vial + tissue + buffer for another 40 seconds.

For Immunocytochemistry: 2-3 hours – Recommended 4% paraformaldehyde/0.05% Glutaraldeyde EM Grade/0.2% picric acid in 0.1M sodium Phosphate buffer, pH 7.3 [Smogyi, P and Takagi. H (1982). Note: The use of picric acid-paraformaldehdye-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neurosciences 7, 1779 – 1783.]

Buffer rinses are needed at least 2 times and 30 minutes each, for all of applications
 Except for the microwave, 2 rinses 3 minutes each is done outside the microwave.

Post Fixation

Conventional: 1 – 2% osmium tetroxide in water or buffer for one hour

For dual LM/LM: post fixation with osmium tetroxide should be avoided.

For Immunocytochemistry: post-osmication or “block staining” should not be carried out. However, post fixing  the tissue with 1% tannic acid is recommended.

Then the “Addition of phosphotungstic acid (PTA) to ethanol for dehydration step, will improve both the ultrastructure and antigenicity of pituitary tissue embedded in LR White.” [Yoko Sakai, Masahiro Hosaka, et al. Histology and Cytology, Vol.68 (2005), No. 5 p.337-34]

Or, post fix in the mix equal volumes of 3% potassium ferricyanide and 2% osmium tetroxide, with a final concentration of 1.5:1  -  buffered or un-buffered, to improved the contrast without using osminum tetroxide [Berryman & Roddewald, 1990. Histochem/cytochem. 38:159 – 70; Berryman et al., 1992. J. Histochem/Cytochem. 40:845-57]

For Microwave: same procedure as the fixation for microwave above.

Dehydration / Resin Infiltration

For best results – the use of a rotator for this step is recommended

Ethanol, 200 proof (EtOH) is the choice of dehydration agent for LW White.
(Acetone acts as a radical scavenger in the resin system and traces of acetone left in the tissue at curing can interfere with polymerization)

Conventional:

For Immunocytochemistry:

Even though the tissue may be taken from 70% ethanol into LW White resin no special procedure is necessary. However, due to the fact that the  nature of the tissue is not always consistent, we feel it is safer with at least one more change with 80% alcohol before going to resin.

 
*  For enhancing contrast: add 2% PTA to this step, if post fix tissue have been treated with 1% TA.
**This step is needed to avoid the tissue shrinkage due to the omitted use of osmium tetroxide during fixation.

For Microwave:

Polymerisation

Anaerobic polymerisation is advised for curing LR White resin.

Thermal (Heat) Cure:  Oven temperature is set at 65°C

If flat embedding is needed for easy orientation of the tissue, we recommend that you should use our PTFE  flat embedding mould instead of silicone. (EMS #70085). Resin must be ‘over-filled’ in  the cavities  then, cut a piece of paraffin,  Aclar film, or polyethylene-based film as big as the mold, and place this film right on top of the resin-filled mould to exclude oxygen.

If BEEM capsules are being used, the lid is removed from the capsule. Filled the capsule and top-off with LR White resin, then use the same method as described above for flat embedding to cover the BEEM capsule.

If gelatin capsules are being used, after fill the capsule with LR White resin, close the capsule and set the temperature at about 55°C (gelatin has a low melting point).

Typical curing time for all the above is overnight to 48 hours, depending on conditions.

Cold cure (use accelerator):

Add 1 drop of the accelerator (10ml bottle, EMS14385) to 10 ml of LR White resin and  mix well. This mixture will polymerise in less than an hour. However this mixture has very poor infiltration. The resin can be cured in gelatin capsules (EMS #70100), BEEM capsule (EMS70020), Moulding cup tray (EMS #70176-10 series) Peel Away Disposable Mould (EMS70180 series).

Microwave Cure:

Use BEEM capsule for microwave curing. In a microwave , LR White resin will polymerise under water.

Load the BEEM capsules into a Capsule Holder (EMS70023). Place the specimen down into the tip of the capsule. Fill the capsules with LR White resin. Put the cap on the capsules (and follow the instruction of this type of capsule holder to secure the capsule lid, so they won’t pop out during the polymerisation process). Place the whole set into the tray (EMS97091), fill the tray with water to cover the capsules (approx 350ml). Place it inside the microwave and start irradiating the resin as follows:

• 10 minutes at a temperature of 60°C* @ full power

• 10 minutes at a temperature of 70°C* @ full power

• 24 minutes at a temperature of 80°C* @ full power

• Let the resin block cool down completely before sectioning.

*Make sure the water is at the requested temperature prior to the run

Trimming and Cutting

When gelatin capsules are used, an initial trimming of the block with a jewelers saw is faster and a final trim with the razor blade is then much easier.

Otherwise, trimming and sectioning of a LR White block may be performed the same way as an  epoxy resin block. Glass knives and  or diamond knives are routinely used. Cutting speed of about 1mm per second with the thickness of 50 – 70nm is suitable.

For LM, sections of 2 – 3µm is nicely obtainable (up to 15 or 20µm if required)

Section Staining

EM – All common stains give good results on tissue embedded in LR White resin.

As an alternative to uranyl acetate, 1% phosphotungstic acid has been proven to be a good general purpose stain, both as a block stain as mentioned earlier , and as a section stain. The use of ethanol and methanol to make up stains should be avoided, because these solvents will soften the cured resin and may remove sections from supporting grids.

IMMUNOCYTOCHEMISTRY – The choice of immunolocalisation technique is entirely up to the user, and PAP, hapten-anti-hapten, avidin-biotin, or gold-colloid methods may be adaptable. (See some helpful references listed below)

Safety

LR White is a low toxicity resin. However all acrylic resins should be considered hazardous. Work under the fume hood, along with safety gloves and goggles are always good laboratory practices. Double-gloves are sometime needed if you are in prolonged direct contact with the methacrylate resins. All chemicals including acrylic resin, should always be tightly closed after each use and stored  in a safe place.

If in case you have direct contact with skin or eyes, wash the affected areas with plenty of water and soap.

References

Fixation

• Ito, S. and Karnovsky, M.J. (1968) Formaldehyde/Glutaraldehyde fixative containing trinitro compounds. J. Cell Biol. 39, 168a -169a.
• McLean I.W. and Nakanem P.K. (1974) Peroxidase-lysin-paraformaldehyde fixative, a new fixative for immoelectron microscopy. J. Histochem, 22, 1077 – 1083
• Smogyi P. and Takagi H. (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlate light and electron microscopic immunocytochemistry. Neurosciences, 7, 1779-1783
• Stefanini, M., De Martino, C and Zamboni, I. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173-174.

Fixation and Embedding in L.R. White

• Newman, G.R. Jasani, B. and Williams, E.D. (1982) The preservation of ultrastructure and antigenicity. J. Microscopy, 127, RP5-RP6.
• Newman, G.R. Jasani, B. and Williams, E.D. (1982) A simple post embedding system for the rapid demonstrationot tissue antigens under the electron microscope. Histochem. J. In press.
• M.A. Hayat. Principles, Methods and applications, Volume 2. , ed. Academic Press, Inc. New York, pp. 38-71.
• J.N. Skepper and J.M. Powell. 2008a.Ultrastructural Immunochemistry (Immunostaining of London Resin(LR) White section for TEM). CSH Protocols doi:1101/pdb.top47 [Abstract/Free Full Text]

Immunostaining

• De May, J., Moeremans, M., Geuens, G., Nuydens, R. and De Brabander, M, (1981). High resolution light and electron microscopic localization of tubulin with the IGS (Immuno gold staining) method. Cell Biol. Int. 5, 889 - 899.
• Hsu, S.M., Raine, L, and Fanger, H. (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577 – 580.
• Jasani, B., Wynford-Thomas, D. and Williams, E.D. (1981) Use of monoclonal antihapten antibodies for immunolocalization of tissue antigens. J. Clin. Path. 34, 1000 – 1002.
• Larson, L. (1979) Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytocheical method. Nature 282, 743 – 746.
• Roth, J. Bendayan, M and Orci, L. (1978) Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.
• Stenberger, L.A. (1972) The unlabelled antibody-peroxidase and the quantitative immunoranium methods in light and electron microscopy. In ‘Techniques of Biophysical and Biochemical Morphology 1’, Rd D.R. Glick and R.M. Rosenbaum. New York, John Wiley and Sons Inc.
• Giberson, et al. 1997. Four-hour processing of clinical/diagnostic specimens for electron microscope . J. Vet. Diagn. Invest. 9:61-67.