258 N
blot detection system Amersham, followed by exposure to ECL HYPER film Amersham.
2.4. Immunohistochemical staining Serial paraffin sections were immunostained according
to the standard streptavidin–biotin peroxidase technique using a Vectastain ABC elite kit Vector Lab., CA. All
sections were treated with 99 formic acid for 3 min, and the sections used for AGE staining were also treated with
0.05 proteinase-K for 30 min. Endogenous peroxidase was inhibited with 0.3 hydrogen peroxidase H O in
Fig. 1. Characterization of the anti-RAGE antibody by Western blotting
2 2
of human brain and bovine lung extracts. Lane 1, anti-RAGE antibody
methanol for 30 min. These sections were also incubated
and immobilized human brain 10 mg lane. Lane 4, anti-RAGE antibody
with 10 horse serum for GFAP, or 10 goat serum
and immobilized bovine lung extract 50 mg lane. Bands are observed at
for Ab, AGE, and RAGE to eliminate nonspecific
approximately 35 kd, 50 kd, and 60 kd in lanes 1 and 4. Lane 2 human
binding. This was followed by incubation overnight at 48C
brain and lane 5 bovine lung show that the immunoreaction was
with the primary antibodies diluted to 1:500|1:1000 in 10
diminished after absorption by the synthesized RAGE peptide. Lanes 3 and 6 show non-immune rabbit IgG and immobilized human brain or
mM PBS pH 7.4. The sections were then sequentially
bovine lung extract, respectively, with no bands being observed. Migra-
incubated with the biotinylated secondary antibody for 1 h,
tion of the protein standards is indicated to the left of lane 1.
with streptavidin–biotin-horseradish peroxidase for 1 h, and with 3,39-diaminobenzidine H O
until the reaction
2 2
products were visualized 1–3 min. Then the sections Western blotting and immunohistochemistry in the same
were counterstained with hematoxylin. Specificity was procedure as described above.
confirmed by applying PBS instead of the primary anti- bodies, or by replacing the primary antibodies with non-
2.6. Semiquantification of RAGE-positive astrocytes immune serum.
The combinations of antibodies used for double staining The number of astrocytes reacting with anti-Ab, AGE or
are summarized in Table 2. We used the anti-GFAP RAGE antibodies was counted in both serial tissue sections
antibody as a marker of reactive astrocytes. It was diluted and double stained sections. In each subject, immuno-
extensively 1:2000–4000, so that observation of im- reactive astrocytes were counted in the hippocampus using
munopositive granules was not prevented.
2
three photomicrographs 1.3 mm each obtained at 3100 magnification.
2.5. Absorption test Specificity of the antibody was verified by absorption
3. Results
tests using Western blotting and immunohistochemistry. Western blotting was performed using anti-RAGE antibody
3.1. Western blot analysis 1:2500 dilution incubated with 10003 molar concen-
tration of synthesized peptides of RAGE residues 167– Immunoblotting was performed to confirm specificity
180, 1 mg ml overnight at 48C. In immunohistochemistry, for RAGE. It demonstrated three major bands at approxi-
anti-RAGE antibody 1:1000 dilution was incubated with mately 35 kd, 50 kd, and 60 kd both in human brain Fig.
10003 molar concentration of above peptides overnight at 1, lane 1 and in bovine lung extract Fig. 1, lane 4. Fig.
48C. These pellets were separated by centrifugation at 1, lanes 2 and 5 shows the result of an absorption test. The
30,000 g for 30 min. This supernatant was used for reaction was disappeared by the preincubation of the
Table 2
a
Antibody combinations for double immunostaining Primary antibody
Secondary antibody Third step
Substrate color Anti-GFAP
mouse IgG APAAP complex
Fast Red red Anti-Ab
biotinylated streptavidin–biotin-
diaminobenzidine Anti-AGE
anti-rabbit IgG horseradish peroxidase
brown Anti-RAGE
a
APAAP: Alkaline phosphatase–antialkaline phosphatase.
N . Sasaki et al. Brain Research 888 2001 256 –262
259
antibody with the synthesized peptides in human brain AGE-positive granules in hippocampal neurons, especially
Fig. 1, lane 2 and bovine lung extract Fig. 1, lane 5. No in CA3 and CA4, than in cortical neurons. These AGE-
immunoreaction was detected with non-immune rabbit IgG positive granules were identified in the presence of NFTs
both in human brain and bovine lung extract Fig. 1, lanes and SPs. In DM and control subjects, the Ab and RAGE
3 and 6. staining patterns were similar to that of AGE, and there
were no remarkable changes compared to the findings in 3.2. Immunohistochemical staining
AD. Fig. 3G–I shows representative astrocytes immuno-
Proteolytic digestion with proteinase-K has been shown stained with anti-Ab G, AGE H, and RAGE I
to expose cross-linked AGE moieties and to enhance AGE antibodies. Astrocytes contained characteristic immuno-
immunoreactivity in the human brain [20]. In the present positive granules [12]. Each immunopositive deposit had a
study, proteinase-K pretreatment enhanced AGE and very fine dott-like structure, and 2–4 mm granules were
RAGE immunoreactivity in astrocytes, but no marked surrounded by these immunopositive structures. Most of
improvement was observed for sections stained with other the granules were located in the cytoplasm of astrocytes,
antibodies. but one of them was attached to the surface of an astrocyte.
In AD brains, NFTs and SPs were strongly positive for Fig. 3J–L shows adjacent serial sections from an AD
the anti-AGE antibody, as has been described elsewhere in brain stained with anti-Ab J, AGE K, and RAGE L
detail [20]. antibodies. Most astrocytes approximately 70–80 con-
There were no or only a few SPs and NFTs in the DM tained both AGE and RAGE-positive granules and their
and control subjects. Numerous GFAP-positive astrocytes distribution was almost the same, while fewer astrocytes
were observed in AD brains, but very few in DM and contained Ab-positive granules approximately 20–30,
control brains. The DM subjects had more AGE-positive arrows.
Ab-J, AGE-K,
and RAGE-L
positive vessels in their brains than the controls. Except for this
granules can be seen to colocalize in one part of the same finding, there were no remarkable differences between DM
astrocytes arrowheads.
Immunopositive astrocytes
and control subjects. characteristically appeared around SP, but AGE- and
Fig. 2 showed the result of an absorption test. Immuno- RAGE-positive astrocytes were also present in areas
reaction in neurons and astrocytes were disappeared by the without SP. In DM and control subjects, AGE- or RAGE-
absorption test Fig. 2B. positive astrocytes were very rare.
AGE-immunopositive granules were observed in AD Fig. 3A, DM Fig. 3B and control brains Fig. 3C.
Many AGE-positive granules were identified in the
4. Discussion
