Assessment of Cell Death: Apoptosis, Necrosis, or In Between - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 15

all other factors being equal, it is usually preferable to use frozen
sections for quantitative analysis between animals.

For IHC staining of brain tissue (or any tissue for that matter), the
type and timing of fi xation can have dramatic effects on immuno-
reactivity and subcellular localization ( 21 ). Verifi cation using
antibodies from different sources as well as co-localization of com-
plementary assessments for a given type of cell death are critical.
The same dilemma holds true for frozen vs. paraffi n-embedded
tissue. To briefl y summarize the benefi ts and issues associated with
either method: Frozen sections are usually better in fl uorescence-
based analysis as the antigens tend to survive the freezing process
better. The formation of ice crystals, however, can disrupt and distort
morphology. Paraffi n embedding is generally more harsh on antigens,
occasionally (not often) rendering some non-immunoreaction,
though morphological resolution is usually excellent. The latter
point is particularly useful when one is employing morphological
assessments.

3.3. Tissue Fixation
and Immunostaining

To determine the suitability of a previously untested antibody for
immuno-staining, it is imperative to fi rst verify that it gives a single
band on a western blot against a suitable homogenate of the tissue
employed as the experimental focus. Although it is possible that
cleavage/minor degradation products of the target antigen can
yield more than one specifi c band, this can be verifi ed using an
appropriate blocking peptide (or, if available, blocking should be
carried out using the full-length target protein). Only when this
type of specifi city test is passed can the investigator be reasonably
confi dent that subsequent immuno-staining signals are an accurate
refl ection of the spatiotemporal expression of the target antigen—
and only the target antigen. There are many parameters that can
affect the outcome of an immuno-staining protocol, and the reader
is advised to consult one of the many excellent resources available,
many through the Internet, for an exhaustive treatment of this
broad subject. In the case of CNS tissue, one should pay particular
attention to issues related to (or potential artifacts arising from)
(1) fi xation, (2) antigen retrieval, (3) blocking steps (e.g., endog-
enous peroxidases), (4) concentration and timing of application
of primary and secondary antibodies that provide the best reso-
lution with the lowest background, (5) washing conditions, and
(6) a development/detection/signaling system appropriate to
the question being asked. With regard to this last point, as the
biochemical and morphological distinctions between different
types of cell death can be very subtle, the advantages offered by
either colorimetric (IHC) or immuno-fl uorescence staining relative
to the desired outcome should be carefully considered from the
very beginning of the experiment. Immunohistochemistry, while
essentially qualitative, is permanent and relatively light-insensitive,

3.4. Testing a New
Antibody