reliability than attempts to count plaque numbers
per square millimeter field (8). Although tangle
density is not formally used in this diagnostic
scheme, tangles in specific regions of the temporal
and occipital lobes are important for the Braak
neuropathologic staging system that correlates with
disease duration (9). Information from both CERAD
and Braak analyses was incorporated into a revised
set of criteria for classifying dementia brains (10).
The need for increased neuropathologic standard-
ization in dementia research has promoted a grow-
ing armamentarium of special stains that are rou-
tinely used in large academic centers to assess
brains from patients who had dementia.
For the practicing pathologist with more limited
laboratory resources, simplifying the diagnostic
evaluation of dementia brains is desirable. Ubiq-
uitin, a small protein tag that marks damaged pro-
teins that are destined for degradation by protea-
somes (11), accumulates in both plaques and
tangles. In addition, ubiquitin is present in Lewy
bodies, a pathologic inclusion that is characteristic
of Parkinson’s disease. Identification of Lewy bod-
ies in patients who are suspected of having Alzhei-
mer’s disease is important, as more than 20% of
pathologically confirmed cases of Alzheimer’s dis-
ease exhibit concurrent Parkinson’s disease pathol-
ogy (12, 13). Moreover, in the absence of
Alzheimer’s-type changes, diffuse Lewy body pa-
thology itself is an alternative cause of dementia.
Considering the ease of our chosen immunohisto-
chemistry technique and its wide availability, we
evaluated the utility of ubiquitin immunostaining
for the neuropathologic diagnosis of patients who
are suspected of having Alzheimer’s disease. We
found that this single immunohistochemical study
evaluated independently of other histologic studies
allowed both general pathologists-in-training and
neuropathologists to render accurate, reproducible
diagnoses.
Ubiquitin Immunohistochemistry
The sections were cut 8 thick from paraffin-
embedded tissue blocks and mounted directly onto
Superfrost/Plus slides (Fisher Scientific, Pittsburgh,
PA). The slides were baked in a 37° C incubator for
12 h before deparaffinization. After the slides were
washed in phosphate-buffered saline (PBS) pH 7.6,
they were pretreated with endogenous peroxide
blocker Endo/Blocker (Biomeda, Foster City, CA).
After a thorough PBS washing and wiping excess
buffer away from the tissue, a prediluted rabbit
polyclonal antibody recognizing human ubiquitin
was applied with a dropper to the tissue. The pri-
mary antibody incubated for 30 min at 37° C in an
incubation chamber before washing in PBS. After
blotting away excess PBS, a prediluted biotinylated
2° was applied with a dropper and incubated for 20
min at 37° C. After another PBS wash, a prediluted
streptavidin conjugated horseradish peroxidase
was applied with a dropper and incubated for 20
min at 37° C. Both prediluted reagents are con-
tained in the Autoprobe III Reagent Sets (Biomeda,
Foster City, CA). Peroxide/diaminobenzidine,3,3Ј-
diaminobenzidine (Sigma, St. Louis, MO) was used
as the chromogen. Nuclei were counterstained with
Biomeda’s aqueous hematoxylin applied with a
dropper for 20 seconds before washing off the ex-
cess in tap water to avoid overstaining. Slides were
blued in ammonical water, then washed in tap wa-
ter before dehydrating and coverslipping. For neg-
ative control sections, nonimmune serum was used
in place of the primary polyclonal antibody.
Scoring of Immunostains
The ubiquitin immunostains were reviewed inde-
pendently by four pathologists using the indicated
template (Fig. 1). (For more details on selection of
appropriate brain sections during gross examina-
tion and assessing these regions by CERAD and
Braak criteria, see Mirra et al. (7) Fig. 1 and Braak
and Braak (9) Fig. 14.) The reviewers—an experi-
enced neuropathologist (CMH), a neuropathology
fellow (CTC), a senior anatomic pathology/clinical
pathology resident (JLC), and a first-year anatomic
pathology resident with brief exposure to autopsy
neuropathology (TJC)—were blinded to age, his-
tory, H&E histology, and original diagnoses during
the semiquantitative scoring of these areas (Fig. 1,
top). The cases were then categorized with respect
to CERAD criteria by factoring in age and history of
dementia, and the Braak stage was assessed using a
simplified algorithm (Fig. 1, bottom). Other patho-
logic features, such as Lewy bodies or vascular
changes, were noted. After 4 to 8 mo, the slides
were scored blindly a second time by the senior
reviewers.
MATERIALS AND METHODS
Pathologic Material
Paraffin blocks were obtained from the Bryan
Alzheimer’s Disease Research Center Brain Bank, a
repository of approximately 900 brains (14). All di-
agnoses in the bank were rendered by an experi-
enced neuropathologist (CMH) based on examina-
tion of hematoxylin and eosin (H&E) stains,
microwave King silver impregnation stains (15),
and Congo red amyloid stains. Tissues from 16 in-
dividuals who exhibited the entire range of
Alzheimer’s-type neuropathology were selected
randomly from each diagnostic category by an in-
dividual not involved in analysis (CR).
Immunohistochemistry for Dementia Diagnosis (C.T. Chu et al.)
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