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We evaluated the expansion of T-lymphocyte subpopulations
following ICH in one brain section per animal at Day1 and Day 10
post-ICH.
The fl uorescence dye-labeled and infrared dye-labeled immu-
nohistochemistry, performed simultaneously, allow comparison of
data acquired under identical conditions such as a tissue processing
and staining. Figure
7
represents an example of immunofl uores-
cence images undertaken at day 1 post-ICH in the area surround-
ing hematoma. The clear increase of double-positive CD3+/CD8+
(T-cytotoxic cells, open white arrows) and single-positive CD8+
2.10. Expected Results
Fig. 7. CD3/CD4 and CD3/CD8 double immunostaining (using fl uorescent dye-labeled secondary antibodies). Micro-
photographs were taken within the vicinity of the hematoma at day 1 post ICH, where
H
defi nes the hematoma. CD3 cells
were immunolabeled with
red
, Alexa Fluor 568, while Alexa Fluor 488 (
green
) labeled CD4 or CD8 cells and DAPI (
blue
)
identifi ed the cell nuclei. (
A
) and (
B
) CD3/CD4 double immunostaining
(A
: ×5,
B
:
×
20 magnifi cation) While CD3+ cells were
abundant (
Aa
,
arrows
in
Ba
) no CD4+ cells (
Ab
) were visible within the entire hemisphere or, at higher magnifi cation, in
area adjacent to ICH perimeter (
Bb
). No CD3+/CD4+ cells were visible in (
Ad
) and (
Bd
). Area bounded by the white square
(
Ad
, ×5) is presented in
Bd
at ×20. Bar 100
μ
m. (
C
) and (
D
) CD3/CD8 double immunostaining (
C
:
×
5;
D: ×
20) In the vicinity
of the ICH injury revealed numerous CD8+ cells (
Cb
,
arrow
in
Db
) were identifi ed as lymphocytes as they colocalized (
Cd
,
arrow
in
Dd
) with the CD3+ cells (
Ca
,
arrow
in
Da
). However, single CD8+ cells were also recognized. An example of sin-
gle- and double-positive CD3/CD8 cells is presented in panel
D
. There are two cells (
ellipse
) that are bothCD3−/CD8+.
While CD3+/CD8+ cells were T-cytotoxic lymphocytes, CD3−/CD8+ cell types most likely are dendritic or natural killer
cells. Low magnifi cation area in white square (
Cd
, ×5) is presented in
Dd
at ×20. Bar 100
μ
m .
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