BOT/MBIO/ZOOL 5364
Materials:
1.
Chuck
(block holder) - round chuck for beam capsules flat chuck for flat embedding
molds
2.
Ultramicrotome
3.
Sharp
razor blades
4.
Specimen
block
5.
Chuck
support (tall for use with ultramicrotome; short for use with binocular
microscope)
Procedure:
A. Trimming Block
1. Anchor specimen block in chuck and screw chuck into
appropriate support. Place under
binocular microscope or in the ultramicrotome.
2. Trim excess resin from the side of the block face. Angle the cuts in order to provide a stable
supported face (50˚) - pyramid shape.
An unstable face will vibrate at high frequency upon impact with the
knife causing "chatter" (periodic variations in thickness).
3. Trim excess resin from the top of the block face. This is only necessary if the tissue is not at the block surface. The more horizontal the cut, the less sectioning will be required to reach a full face.
4. Trim the block face to form a broad trapezoid, the top edge
being smaller than the bottom. Avoid
cutting away any tissue. The block face
may be large at this stage, but preferably less than 1 mm.
The advantages of the
trapezoid block face are:
a.
Sectioning
from wider to narrower means the sectioning is less wearing to the knife as the
cut is made.
b.
Forms
ribbons more easily if top and bottom are exactly parallel.
c.
Easy
to orient direction of sectioning in the microscope. The block will be in the microtome at the same angle each time it
is sectioned (top is smaller).
d.
Provides
orientation to the tissue.
If the top and bottom are
not parallel, the block will not ribbon or the ribbon will curve. This will also wear out the knives unevenly.
Use a new, clean razor blade
for the final trimming. This may also
be done with a glass knife on a microtome.
a. A dirty or
unsmooth lower edge will not contact the knife properly.
b. A dirty upper
edge may cause the section not to detach from the block face and therefore be
dragged back over the knife edge.
B. Facing
the Block
1.
Face the block using a
glass knife (a boat does not need to be present and may interfere). The microtome pivot control should be
reset. Advance the knife using the
manual advance, but use the motor to move the specimen arm. If the knife becomes dull, change knives.
2.
Reshape the block face
into a trapezoid using a new, clean, sharp razor blade so that the top and
bottom are parallel and the surface is glass-like. There is a limitation of the width of the block as to available cutting
area on the glass knife (approximately the left 1/2 of the knife is suitable
for sectioning). Larger sections are
exceedingly difficult to obtain.
C. Thick
Sectioning
1.
Use a glass knife with
a distilled water-filled boat using a syringe (be careful with the needle and
keep it capped when not in use). Make sure pivot control is set appropriately
(10 nm is our default setting).
2.
Cut thick
section(s). Remove sections from the
boat with an empty slotted grid, clean wire loop or wooden spatula.
3.
Transfer the drop
containing the thick section(s) to a drop of water on a clean glass slide. Lift the transfer device away slowly. The section should be floating in the drop. Pretreating slides by dipping them in a warm
solution of 0.5% gelatin in distilled water and letting them dry prior to use
will make the drop “bead up” and permanently adhere the section to the slide.
4.
To accelerate adherence
of the section, place the slide on a low temperature hot plate. This will relax and flatten the section and
evaporate water allowing attachment of the section(s) to the slide. This may take approximately 2 minutes or so.
5.
Optionally, you may
choose to stain the section if phase or other contrast microscopy are
unavailable. To stain the material,
place a drop of stain (toluidine blue, or methylene blue) on section.
Zoologists frequently use a mixture of methylene blue and basic fuchsin, called
Paragon.
6.
Warm carefully until the
edges of the drop begin to dry (30-60 sec).
Remove before boiling or the section will be folded.
7.
Remove from warmth;
rinse gently with distilled water; dry.
8.
View with light
microscope. If you want to increase
contrast, used phase or interference contrast microscopy or stain the material. (Staining may be repeated to increase density.)
9.
It is possible to use
oil immersion if desired. For a
temporary slide, mount cover slip with glycerol (removable with water). For a permanent slide, seal the edges of the
cover slip with epoxy glue. ("Permount"
causes the stain to bleach out over an extended period of time and is not
recommended.)