Effects of maternal obesity on the hippocampal
vascularization and coverage of capillaries by end-feet of
astrocytes in offspring: no significant difference in the length
density of CD31 + capillaries between two groups of mice (
A ); an example of the length density of AQP4 + capillaries
measurement. The green lines (arrow) represent the
intersection between isotropic virtual plane and the focal
plane. Capillaries that are in focus and intersected by virtual
planes are counted as positive AQP4.For estimating the
reference volume, we use the four corner points of the
box.Scale bar = 20 um ( B ); a significant higher coverage of
capillaries with end-feet of astrocytes ( A ) without changes
in vascularization in offspring of obese dams ( C ); a
significant positive correlation between length density of
AQP4 + capillaries and number of astrocytic branches ( D );
a significant higher endothelial expression of LCN2,
suggesting longer LCN2 + capillaries, in the hippocampus of
offspring of obese dams ( E ); examples of hippocampal
endothelial LCN2 expression in the offspring of control ( F )
and obese dams ( G ). Scale bar = 20 um; a significant
positive correlation between length density of LCN2 +
capillaries and number of astrocytic branches ( H ); a
significant negative correlation between length density of
LCN2 + capillaries and volume of hippocampus ( I ). * p
T cells, B cells and NK cells are not involved in the benefit of
FPR2 antagonism in NMOSD mice. ( A ) 9.4T-T2 weighted MR
images showing brain lesions within the axial position in
RAG2 -/- NMOSD mice treated with vehicle or Quin-C7 (32
mg/kg/day). ( B ) Bar graph showing the volumes of
intracranial lesions in RAG2 -/- NMOSD mice treated with
vehicle or Quin-C7 (32 mg/kg/day). ( C ) Schematic diagram
depicts experimental design. NMOSD was induced in NK
depleted mice by intracerebral injection of AQP4-IgG with
hC. Thereafter, these mice received daily administration of
Quin-C7 (32 mg/kg) or an equal volume of vehicle for 4
consecutive days starting immediately after NMOSD
induction. On day 4 after NMOSD, the imaging analysis was
performed. For NK cell depletion, mice were injected i.p. with
250 ug anti-NK1.1 mAb or isotype control every three days,
the first time was injected one day before NMOSD induction.
( D-E ) Flow cytometry gating strategy ( D ) and
quantification of NK cell percentage ( E ) in the peripheral
blood of mice with or without anti-NK1.1 mAb treatment. n =
6 per group. ( F ) Representative 9.4T-T2 weighted MR
images showing brain lesions within the axial position in
indicated groups of NMOSD mice. ( G ) The histogram shows
the volumes of intracranial lesions in indicated groups of
NMOSD mice. Data are presented as mean ± SEM. ** P <
0.01.Index in PubMed under a CC BY license. PMID:
40225578
Microglia contribute to the benefit of FPR2 antagonism in NMOSD mice. ( A ) Schematic diagram illustrates drug
administration and experimental design. C57BL/6 mice
received oral gavage of PLX5622 for 14 days and
continuously until the experiment ended. NMOSD was
induced in PLX5622-treated mice by intracerebral injection
of AQP4-IgG with hC. Thereafter, these mice received daily
administration of Quin-C7 (32 mg/kg) or an equal volume of
vehicle for 4 consecutive days starting immediately after
NMOSD induction. On day 4 after NMOSD, the imaging
analysis was performed. ( B-C ) Flow cytometry gating
strategy ( B ) and bar graph ( C ) showing microglia
percentage in the brain tissues obtained from mice receiving
a control diet or PLX5622 for 14 days. n = 6 per group. ( D )
9.4T-T2 weighted MR images showing brain lesions within
the axial position in indicated groups of NMOSD mice. ( E )
Bar graph showing the volumes of intracranial lesions in
indicated groups of NMOSD mice. Data are presented as
mean ± SEM. ** P < 0.01.Index in PubMed under a CC BY
license. PMID: 40225578
FPR2 antagonism reduces NMOSD pathology in mice. ( A )
Schematic diagram depicts experimental design. Mice
received daily gavage administration of different dosages of
Quin-C7 or an equal volume of vehicle for four consecutive
days, starting immediately after NMOSD induction. Mouse
brain tissue was collected for immunostaining analysis on
day 4 post-NMOSD. ( B ) Immunostaining shows the
expression of AQP4, GFAP, and MBP in indicated groups of
NMOSD mice, white lines represent the area with the loss of
AQP4, GFAP and MBP. Scale bar: 1mm. ( C ) Bar graphs
showing the loss of AQP4, GFAP and MBP. n = 8-11 mice per
group. ( D ) 9.4T-T2 weighted MR images showing brain
lesions within the axial position in vehicle or Quin-C7-treated
(32 mg/kg/day) NMOSD mice on day 4. ( E ) Bar graph
showing the intracranial lesion volumes of the vehicle or
Quin-C7-treated (32 mg/kg/day) NMOSD mice. Data are
presented as mean ± SEM. * P < 0.05, ** P < 0.01.Index in
PubMed under a CC BY license. PMID: 40225578
Western blot analysis of Aquaporin 4 using anti-Aquaporin 4
antibody (MBS178038). Electrophoresis was performed on a
5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving
gel) for 2-3 hours. The sample well of each lane was loaded
with 30 ug of sample under reducing conditions. Lane 1: rat
brain tissue lysates, Lane 2: rat brain tissue lysates, Lane 3:
mouse brain tissue lysates, Lane 4: mouse brain tissue
lysates. After electrophoresis, proteins were transferred to a
nitrocellulose membrane at 150 mA for 50-90 minutes.
Blocked the membrane with 5% non-fat milk/TBS for 1.5
hour at RT. The membrane was incubated with rabbit anti-
Aquaporin 4 antigen affinity purified polyclonal antibody
(Catalog # MBS178038) at 0.5 ug/mL overnight at 4°C, then
washed with TBS-0.1%Tween 3 times with 5 minutes each
and probed with a goat anti-rabbit IgG-HRP secondary
antibody at a dilution of 1:5000 for 1.5 hour at RT. The
signal is developed using an Enhanced Chemiluminescent
detection (ECL) kit (Catalog # Please Inquire) with Tanon 5200
system. A specific band was detected for Aquaporin 4 at approximately 37 kDa. The expected band size for
Aquaporin 4 is at 35 kDa.
Western blot analysis of AQP4 using anti-AQP4 antibody
(MBS178038). Electrophoresis was performed on a 10% SDS-
PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2
hours. The sample well of each lane was loaded with 30 ug
of sample under reducing conditions. Lane 1: Zebrafish head
tissue lysates. After electrophoresis, proteins were
transferred to a nitrocellulose membrane at 150 mA for
50-90 minutes. Blocked the membrane with 5% non-fat
milk/TBS for 1.5 hour at RT. The membrane was incubated
with rabbit anti-AQP4 antigen affinity purified polyclonal
antibody (MBS178038) at 0.5 ug/ml overnight at 4°C, then
washed with TBS-0.1%Tween-20 3 times with 5 minutes
each and probed with a goat anti-rabbit IgG-HRP secondary
antibody (Catalog # MBS176703) at a dilution of 1:5000 for 1.5
hour at RT. The signal is developed using an ECL Plus
Western Blotting Substrate (Catalog # MBS1753888) with
Tanon 5200 system. A specific band was detected for AQP4
at approximately 37 kDa.The expected band size for AQP4 is
at 37 kDa.
IHC analysis of Aquaporin 4 using anti-Aquaporin 4 antibody
(MBS178038). Aquaporin 4 was detected in a paraffin-embedded
section of human lung cancer tissue. Heat mediated antigen
retrieval was performed in EDTA buffer (pH 8.0, epitope
retrieval solution). The tissue section was blocked with 10%
goat serum. The tissue section was then incubated with 2
ug/ml rabbit anti-Aquaporin 4 Antibody (MBS178038) overnight
at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used
as secondary antibody and incubated for 30 minutes at
37°C. The tissue section was developed using HRP
Conjugated Rabbit IgG Super Vision Assay Kit (Catalog #
Please Inquire) with DAB as the chromogen.
IHC analysis of Aquaporin 4 using anti-Aquaporin 4 antibody
(MBS178038). Aquaporin 4 was detected in a paraffin-embedded
section of mouse brain tissue. Heat mediated antigen
retrieval was performed in EDTA buffer (pH 8.0, epitope
retrieval solution). The tissue section was blocked with 10%
goat serum. The tissue section was then incubated with 2
ug/ml rabbit anti-Aquaporin 4 Antibody (MBS178038) overnight
at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used
as secondary antibody and incubated for 30 minutes at
37°C. The tissue section was developed using HRP
Conjugated Rabbit IgG Super Vision Assay Kit (Catalog #
Please Inquire) with DAB as the chromogen.
IHC analysis of Aquaporin 4 using anti-Aquaporin 4 antibody
(MBS178038). Aquaporin 4 was detected in a paraffin-embedded
section of rat brain tissue. Heat mediated antigen retrieval
was performed in EDTA buffer (pH 8.0, epitope retrieval
solution). The tissue section was blocked with 10% goat
serum. The tissue section was then incubated with 2 ug/ml
rabbit anti-Aquaporin 4 Antibody (MBS178038) overnight at 4°C.Peroxidase Conjugated Goat Anti-rabbit IgG was used as
secondary antibody and incubated for 30 minutes at 37°C.
The tissue section was developed using HRP Conjugated
Rabbit IgG Super Vision Assay Kit (Catalog # Please Inquire) with
DAB as the chromogen.
IHC analysis of Aquaporin 4 using anti-Aquaporin 4 antibody
(MBS178038). Aquaporin 4 was detected in frozen section of
Mouse Brain Tissue. The tissue section was blocked with
10% goat serum. The tissue section was then incubated with
1ug/ml rabbit anti-Aquaporin 4 Antibody (MBS178038) overnight
at 4°C. Biotinylated goat anti-rabbit IgG was used as
secondary antibody and incubated for 30 minutes at 37°C.
The tissue section was developed using Strepavidin-Biotin-
Complex (SABC)(Catalog # Please Inquire) with DAB as the
chromogen.
IHC analysis of Aquaporin 4 using anti-Aquaporin 4 antibody
(MBS178038). Aquaporin 4 was detected in frozen section of Rat
Brain Tissue. The tissue section was blocked with 10% goat
serum. The tissue section was then incubated with 1ug/ml
rabbit anti-Aquaporin 4 Antibody (MBS178038) overnight at 4°C.
Biotinylated goat anti-rabbit IgG was used as secondary
antibody and incubated for 30 minutes at 37°C. The tissue
section was developed using Strepavidin-Biotin-Complex
(SABC)(Catalog # Please Inquire) with DAB as the chromogen.
Western blot analysis of Aquaporin 4 using anti-Aquaporin 4
antibody (MBS178038). Electrophoresis was performed on a
5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving
gel) for 2-3 hours. The sample well of each lane was loaded
with 30 ug of sample under reducing conditions. Lane 1:
Normal group-rat colon tissue lysates, Lane 2: Model group-
rat colon tissue lysates, Lane 3: Traditional Chinese
medicine-rat colon tissue lysates, Lane 4: Western medicine-
rat colon tissue lysates. After electrophoresis, proteins were
transferred to a nitrocellulose membrane at 150 mA for
50-90 minutes. Blocked the membrane with 5% non-fat
milk/TBS for 1.5 hour at RT. The membrane was incubated
with rabbit anti-Aquaporin 4 antigen affinity purified
polyclonal antibody (Catalog # MBS178038) at 1:1000 overnight
at 4°C, then washed with TBS-0.1%Tween 3 times with 5
minutes each and probed with a goat anti-rabbit IgG-HRP
secondary antibody for 1 hour at RT. The signal is developed
using an Enhanced Chemiluminescent detection (ECL) kit
(Catalog # Please Inquire) with ChemiDoc MP system. A specific
band was detected for Aquaporin 4 at approximately 37 kDa.
The expected band size for Aquaporin 4 is at 35 kDa.
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