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Using L.R. White for Hard Tissue

Using LR White for hard tissue. A user note for C023, C024, C025, C026, C027.

L.R. White can be used for the microtomy of decalcified bone and teeth and also for microtomy or "sawing and grinding" of undecalcified tissues.

DECALCIFIED TISSUE:

May be processed, cut and stained similarly to soft tissue (see Using L.R. White for Light Microscopy), except that dehydration and infiltration times may need to be extended depending on the size of tissue. It is also recommended that bone be "de-fatted" to improve the penetration of resin into marrow cavities. This can be achieved by using chloroform after dehydration and returning to absolute alcohol to remove the chloroform before infiltrating with resin and polymerising.

UNDECALCIFIED TISSUE:

Dehydration and infiltration times will vary depending on size and density of tissue. Those laboratories using Methyl or Butyl methacrylate at present can use similar dehydration times, but infiltration will probably be shortened due to the low viscosity of the resin.

DEHYDRATION:

A graded series of alcohol should be used for dehydration of tissue, and when processing bone "de-fatting" is recommended to improve the penetration of resin into marrow cavities. This can be done using chloroform for the same length of time that would be necessary to clear the tissue. The bone should then be taken back to absolute alcohol and given sufficient changes to remove the chloroform before infiltration with L.R. White (Hard Grade).

INFILTRATION:

Several changes of resin will be necessary and impregnation under vacuum is recommended.

POLYMERISATION:

The tissue can be heat or accelerator cured after embedding in strong plastic moulds, such as JB4 or Peel-a-way type, or aluminium foil dishes.

When heat curing the moulds should first be filled with resin then the tissue added and oriented. Polymerisation will occur in 18-24 hours at 60 degrees C 2 degrees C. The surface of the block exposed to oxygen may remain slightly sticky, but this will not affect the cutting quality of the face of the block. Some ovens are not capable of controlling temperature so closely and if faced with overly brittle blocks, this is the first parameter to check.

When accelerator or 'cold' curing the moulds should be placed in a bath of ice-cold water to disperse the heat produced during the exothermic polymerisation. The base of the moulds should be smeared with accelerator using a cotton-wool bud or swab, the accelerator is then added to the resin, 1 drop per 10 ml resin, and thoroughly mixed before pouring into the mould. The tissue is then placed into the mould and oriented. Polymerisation should occur in 10-20 minutes. If it occurs faster than this, we recommend either more careful metering of the one drop of accelerator or a higher volume of resin per drop of accelerator. N.B. the accelerator does have some toxic risk and contact with skin and eyes should be avoided.

CUTTING AND MOUNTING:

Bone marrow trephines and small pieces of cancellous bone may be cut using a motorised heavy duty microtome, but larger pieces of cancellous bone, cortical bone and teeth offer too much resistance to the microtome knife and preparations of this material must be prepared by sawing and grinding.

MICROTOMY:

Sections can be cut, using Ralph type glass knives for trephines or a tungsten carbide knife for larger pieces of cancellous bone, from 2-10 micron. Blocks can be cut dry, the sections picked up and floated out on a hot plate at 60 - 70 degrees C using the following solution: to 20 ml acetone add 0.5 ml benzyl alcohol mix then make up to 50 ml with distilled water. A section adhesive such as egg albumin can be added to this if required. Sections should be allowed to dry on the hot plate for at least 30 minutes before staining.

STAINING AND GRINDING:

Thick slices 150-200 micron can be cut using a milling machine and then ground to the required thickness, usually 20 micron for staining or 70 micron for microradiography; the section is inclined to fragment if grinding is continued much below 20 micron.

Using the newer types of saw microtome, such as the Leitz 1600, which has a diamond-coated-internal-hole-saw, sections can actually be cut at 20 micron and no further grinding is necessary.

SECTION STAINING:

Sections of material embedded in L.R. White are stained "free floating". Times of staining are usually longer than those for paraffin sections, and dehydration through alcohol should be avoided. A recommended schedule for Haematoxylin and Eosin staining is as follows:

  1. Remove Ca deposits, which would otherwise interfere with the staining, by treating with Kristensene's Decalcifying Solution for about 15 minutes.
  2. Wash in running tap water for a few minutes to remove the formic acid from the tissue.
  3. Transfer the section to several changes of distilled water, a few minutes each, and then into a 0.5% w/v solution of periodic acid in distilled water where it is left for 5 minutes.
  4. Wash the section to several changes of distilled water and stain with Harris Haematoxylin for about 1 hour.
  5. Transfer the section, after a short time in distilled water, into running tap water to "blue" the Haematoxylin stained tissue.
  6. Check the Haematoxylin stain; if the tissue should be overstained or the surface of the resin has become stained with Haematoxylin, this may be corrected by a short differentiation in acid alcohol (0.5% HCl in 70% ethanol) and "reblueing" of the stained tissue.
  7. Rinse the section in distilled water and counterstain it, using a 5% solution of Eosin Y in distilled water; leave the section in the Eosin stain for 30 minutes to 1 hour, wash briefly in running water and check the staining of the tissue. Nuclei and haematoxophilic elements should be bright blue, cytoplastic structures in various shades of red-pink.
  8. Rinse the section in distilled water, blot dry with filter paper and either clear briefly in xylene and mount in DPX, or mount directly in L.R. White resin by adding a drop of accelerator to 1 ml of resin.
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