TRAINING PROGRAM
355
20 minute changes of styrene if a polyester embedment is to be employed
(Chapter 4.2 and 3).
4. Plan to embed in either Araldite (Chapter 4.10) or Vestopal W
(Chapter 4.13). Epon can be employed as a substitute but there are pitfalls in its use (Chapter 4.11). Make up about 100 ml of the resin of
choice with its appropriate hardeners and accelerators.
5. Infiltrate with the resin mixture, allowing adequate time for this
(Chapters 4.4 and 5). If the tissue blocks are as small as they should be,
begin with a 30 minute exposure on a mechanical shaker to a resin
mixture so diluted with its appropriate solvent (propylene oxide or
styrene) that it is quite watery. This will mean about a two-thirds dilution with Araldite or a one-half dilution with Vestopal W. Follow this
with another 30 minute exposure, accompanied by shaking, to a resin
mixture having about one-half the previous dilution. It should be of
such a viscosity that it barely sloshes as it is shaken. Finally expose the
tissue for 1 hour to the undiluted resin mixture, with occasional stirring.
6. Transfer tissue blocks to fresh embedding medium in individual
gelatin capsules (Chapter 4.4). This can be done best with a toothpick.
7. Polymerize in a 60°C oven. Allow 2 days for Araldite (Chapter 4.10),
1 day for Vestopal W (Chapter 4.13).
8. Trim blocks as truncated pyramids with a face no more than 0.5 mm
square (Chapter 4.19).
9. Prepare a clean glass knife with a gross angle of no more than 45°
(Chapter 5.13-15), and mount a trough upon it (Chapter 5.17). Orient
in the microtome so that the front face of the knife is tipped toward the
block about 3-4° (Chapter 5.18).
10. Mount trimmed block in microtome and face with old knife or
with undesirable part of new knife (Chapter 5.21). Then move good
knife edge into position and cautiously advance until contact is made
with the block by cutting a section, or a part of one. Fill the trough with
10% acetone to wet the knife edge (Chapter 5.20).
11. Section with the intention of getting sections about 600 A thick
(Chapter 5.21). Sections which are silver or very pale gold will prove most
generally useful (Chapter 5.23).
12. Pick up sections directly upon naked 200 mesh (or smaller) grids
by touching floating ribbons with dry grids (Chapter 5.25).
13. "Stain" Araldite sections for 30 minutes, or Vestopal W sections
for 10 minutes, with Reynolds alkaline lead citrate (Chapter 7.3).
14. Compare results after the various fixatives. The tissue fixed first
355
20 minute changes of styrene if a polyester embedment is to be employed
(Chapter 4.2 and 3).
4. Plan to embed in either Araldite (Chapter 4.10) or Vestopal W
(Chapter 4.13). Epon can be employed as a substitute but there are pitfalls in its use (Chapter 4.11). Make up about 100 ml of the resin of
choice with its appropriate hardeners and accelerators.
5. Infiltrate with the resin mixture, allowing adequate time for this
(Chapters 4.4 and 5). If the tissue blocks are as small as they should be,
begin with a 30 minute exposure on a mechanical shaker to a resin
mixture so diluted with its appropriate solvent (propylene oxide or
styrene) that it is quite watery. This will mean about a two-thirds dilution with Araldite or a one-half dilution with Vestopal W. Follow this
with another 30 minute exposure, accompanied by shaking, to a resin
mixture having about one-half the previous dilution. It should be of
such a viscosity that it barely sloshes as it is shaken. Finally expose the
tissue for 1 hour to the undiluted resin mixture, with occasional stirring.
6. Transfer tissue blocks to fresh embedding medium in individual
gelatin capsules (Chapter 4.4). This can be done best with a toothpick.
7. Polymerize in a 60°C oven. Allow 2 days for Araldite (Chapter 4.10),
1 day for Vestopal W (Chapter 4.13).
8. Trim blocks as truncated pyramids with a face no more than 0.5 mm
square (Chapter 4.19).
9. Prepare a clean glass knife with a gross angle of no more than 45°
(Chapter 5.13-15), and mount a trough upon it (Chapter 5.17). Orient
in the microtome so that the front face of the knife is tipped toward the
block about 3-4° (Chapter 5.18).
10. Mount trimmed block in microtome and face with old knife or
with undesirable part of new knife (Chapter 5.21). Then move good
knife edge into position and cautiously advance until contact is made
with the block by cutting a section, or a part of one. Fill the trough with
10% acetone to wet the knife edge (Chapter 5.20).
11. Section with the intention of getting sections about 600 A thick
(Chapter 5.21). Sections which are silver or very pale gold will prove most
generally useful (Chapter 5.23).
12. Pick up sections directly upon naked 200 mesh (or smaller) grids
by touching floating ribbons with dry grids (Chapter 5.25).
13. "Stain" Araldite sections for 30 minutes, or Vestopal W sections
for 10 minutes, with Reynolds alkaline lead citrate (Chapter 7.3).
14. Compare results after the various fixatives. The tissue fixed first