Histopathological Assessments of Animal Models of Cerebral Ischemia - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 7

2. Clearing: During this step, the dehydrating agent from the
tissue is replaced by clearing agent. Fluids which are miscible
with both dehydrating agents and paraffi n wax are generally
hydrocarbon solvents (with refractive index similar to protein)
such as xylene, toluene, chloroform, or citrus fruit oils (com-
monly used now owing to its non-toxic nature). When tissue
processing is carried out manually, one can observe that tissue
appears translucent once the dehydrating agent is completely
replaced by clearing agent.
3. Impregnation: During this step, the clearing agent is replaced
by an embedding medium; i.e., paraffi n wax. In an automated
tissue processor, tissue is processed until this step. Usually for
tissues like brain, an overnight (Table 1 ) program in the auto-
mated tissue processor is used. At the end of a paraffi n wax
cycle, the basket containing tissue cassettes is taken out and
individual cassettes are immersed in melted wax chamber of an
embedding center (Fig. 3 ).
4. Embedding tissue in paraffi n wax: During this step, individual
cassettes are open and the bottom/larger part of a cassette is
used as chuck to mount the tissue. The most important point
in mounting the piece of brain on a chuck is its orientation.

3.3. Cutting of Brain
Sections

For this task, one is required to have practical experience under the
supervision of a skilled teacher. In particular, one must learn how
to adjust microtome settings, trim tissue blocks, and select the
knife angle. Coronal brain sections of 5-10 mm can be sectioned
with a microtome (Leica Microsystems GmbH, Ernst-Leitz-Strasse
17-3735578, Wetzlar). Cut sections are placed in a water bath
maintained at 10°C below the melting point of the paraffi n wax.
The purpose of placing sections in a water bath is to remove any
folds in tissue sections that originated at the time of cutting. The
section should be laid shiny side down (dull side up) for 30-40 s
(time enough to fl atten the section; a longer time will expand the
section excessively and destroy its tissue structure). Sections are
taken up on to the slides and the slides are labeled appropriately
using a pencil or permanent marker pen which is resistant to solvents
(important as the staining steps involve solvents). Excessive water
from the slide holding the tissue sections should be removed using
a paper tissue wipe. Slides should be placed in a metal slide rack and
sections on the slide should be allowed to dry at room temperature
for some time, followed by storage in a 37°C oven overnight.

3.4. Hematoxylin
and Eosin Y Staining
of Paraffi n Brain
Sections

Following is the step-by-step process to stain sections.

1. Dewax sections either using xylene or citrus fruit oil.
2. Hydrate sections through different grades (100, 95, and 70%)
of ethanol to water. Two minutes for each step is enough.