Biomedical Engineering Reference
In-Depth Information
By placing a mark on the superior aspect of the albino rat globe and a
mark on the optic nerve, it is possible to get uniform vertical midsag-
ittal sections. With these uniform sections, layers of the sensory retina
(e.g., outer nuclear layer) may be measured at set intervals and spider
graphs can be created [
36
,
78
,
87
].
After the globe or extraocular tissue is trimmed, the tissue is pro-
cessed which involves the removal of water from the tissue and
infiltration with paraffin wax [
11
,
77
,
84
,
88
,
89
]. After processing
is complete the tissues are embedded in molds filled with paraffin
wax. The globes are oriented in the same direction as the mold. The
histotechnologist must ensure that the lens (especially of larger
globes) comes in complete contact with the bottom of the mold;
otherwise the posterior portion of the lens will be absent (Fig.
1c
).
2.7.4 Paraffin Embedding
and Processing of Ocular
Tissues
After the paraffin in the molds hardens, ribbons of tissue sections in
paraffin are cut from the face of the block by using a microtome
(Fig.
1c
)[
11
,
13
,
89
]. The most common difficulty in sectioning
the globe is getting good sections of the lens. There are various
techniques used by histotechnologists, such as cooling the block
face with ice or applying a substance, that will soften the lens and
allow for better ocular sections. After a ribbon has been obtained, it
is floated on a water bath. The temperature of the water bath is
important. It should not be too warm because the globe will
expand quickly and not too cool because the ocular section will
not expand enough and artifacts will be present. The ocular section
is picked up from the water bath with a glass slide. Adherence to the
slide is assisted by applying an adhesive (e.g., poly-
L
-lysine) to the
water bath. After the section is on the slide, there is a drying period
before staining. Temperature during drying is important to ensure
good adhesion of the section to the slide. In addition to the
standard stain combination of hematoxylin and eosin, a silver
stain may be used for examining axons; luxol fast blue may be
used for examining myelin and periodic acid Schiff helps in the
examination of Descemet's membrane, the corneal endothelium,
and lenticular capsule.
2.7.5 Sectioning and
Staining Paraffin-
Embedded Ocular Tissue
2.8 Extraocular
Tissue Evaluation
Microscopic evaluation of the extraocular tissues involves the same
techniques used for the microscopic examination of routine tissues
evaluated in toxicity studies. It is important to clearly label the
tissue, being as specific as possible, and to indicate if the tissue is
from the right or left eye.
