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Fig. 6. CD3/CD4 and CD3/CD8 double immunostaining using near infrared dye-labeled secondary antibodies. The infrared
signal intensities of CD3+ (wavelength 700 nm,
red
) and CD4+ or CD8+ cells (wavelength 800 nm,
green
) were quantifi ed
in fi ve region of interests (ROIs,
white squares
) in one brain section per animal at each time point. To maintain analyzed
areas identical in both (700 nm and 800 nm) channels, ROIs were drawn on the merged
red
and
green images
. (
A
) and (
B
)
Example of scanned ipsilateral brain sections. These images were acquired using identical scanning parameters. The
bright visualized CD3+ cell signals at day 1 had reduced intensity at day 10 post-ICH (
red
in
A
and
B
). While at day 1 CD4+
signals were not recognizable, they became marginally brighter at day 10 (
green, upper panel
in
A
and
B
). The low intensity
CD8+ cell signals remain unchanged at both examined time points (
green
,
lower panel
in
A
and
B
). (
B
) and (
D
) The graphi-
cal illustrations of imaging acquired data. The numerical (Raw Integrated Intensities, arbitrary units (AU)) intensity data are
presented in Table
6
and summarized in (
C
) and (
D
). (
C
) At day 1, CD3+ cell signal intensities were dramatically increased
in ipsilateral hemisphere without observable differences in CD4+ and CD8+ signal intensities. In the contralateral hemi-
sphere, signal intensities of CD3+ and CD8+ cells were higher compared to CD4+ cells. There was a signifi cant increase
in CD marker intensities in the contralateral hemisphere compared to ipsilateral at day 1. (
D
) At day 10, the signal intensity
of CD3+ cells remain elevated compared to CD4+ and CD8+ cells in both ipsi- and contralateral hemispheres (*
p
< 0.001
vs. CD8 and CD4; #
p
< 0.01 vs. CD4; **
p
< 0.05 vs. ipsilateral hemisphere; ANOVA).
parameters recorded in these reports. Dependent on the study
focus, choose an appropriate parameter for image analysis. For
our study, the “Raw Signal Intensities” were used.
5. For data correction, subtract the averaged negative control sig-
nal intensities from signal intensities of each corresponding
hemisphere in each channel. Both real and corrected data are
presented in Table
6
. The fl uorescence signal intensity is
expressed in integrated-intensity units (arbitrary units, AU)
(Fig.
6c
,
d
).
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