Page 85 - JCTR-11-4

Journal of Clinical and
Translational Research NADPH oxidase inhibition in a rodent stroke model

staining (20 µM; 1:20 series). Slices were obtained from Exciter Wide Field Microscope, Zeiss, Germany), and
striatal (morphology from approximately +2 mm to analysed using the MicroManager v1.4.21 software
−0.2mm relative to the anterior bregma) and hippocampal with an attached charge-coupled device camera (Retiga
(morphology from approximately −1.45mm to −3.5mm R1, QImaging, USA), utilising filter cubes suitable for
relative to the anterior bregma) regions, stored in 0.02% 400 nM (blue) and 546 nM (red) emission imaging.
sodium azide and Tris-buffered saline (TBS) solution, and Region-specific images were obtained from both ipsilateral
sealed at 4°C until required. For Nissl staining, 20 µM and contralateral hemispheres with three images/slice/
slices were taken throughout the cortex and mounted onto hemisphere from the striatum (STR), sensorimotor cortex
glass slides (10149870, Fisher, UK), air-dried and stored at (SC), and hippocampal CA1 (CA1) regions, and one
−80°C until required. image/slice/hemisphere from the dentate gyrus hilus (DG)
region. NeuN cells were manually counted within the
+
2.11. Immunofluorescence staining and imaging central diagonal of a 4 × 4 grid overlaid on each image.
Immunofluorescence staining was performed using a Counts from replicate images were averaged per slice. Slice
free-floating method to identify neuronal nuclei (NeuN; averages were summed across all slices to represent the
neuronal cell marker), BrdU (cell proliferation marker), total NeuN cell count per region.
+
and cluster of differentiation marker 31 (CD31; vascular Neuronal nuclei + BrdU and CD31 + DAPI
endothelial cell marker). 4’,6-diamidino-2-phenylindole immunofluorescent images were acquired using laser
nuclear DNA stain (DAPI; cell nucleus marker) was also scanning confocal microscopy (Zeiss LSM880C, Zeiss,
used. See Table A1 for antibody details. For NeuN DAPI Germany) and analysed using the Zen Imaging software
+
staining, six randomly selected brains (three per treatment (Black v2.3). NeuN BrdU images were obtained from
+
group) were stained, and for both CD31 and NeuN BrdU the DG region at 10× and 63× magnification. Whole
+
staining, 10 randomly selected brains (five per treatment DG images were taken at 10× for both the ipsilateral and
group) were stained. All immunofluorescence staining contralateral hemispheres, one image/slice/hemisphere.
steps were performed at room temperature unless otherwise BrdU stained cells were manually counted for each
+
stated. For NeuN DAPI and CD31 staining, sections were image within the granule cell layer and subgranular
+
washed in TBS (3 times for 5 min each), blocked and zones. Total BrdU cell count per hemisphere was used for
+
permeabilised in blocking buffer (1 h; 5% normal goat analysis. For NeuN /BrdU co-localisation, 50 randomly
+
+
serum + 0.3% Triton X-100 and TBS). Sections were then selected BrdU cells per animal (25 per hemisphere)
+
incubated overnight at 4°C with primary antibodies. After were imaged using 63× magnification Z-stacks (2 µM Z
washing with TBS (3 times for 5 min each), sections were plane increments) within the DG granule cell layer and
incubated with secondary antibodies for 2 h, followed subgranular zone. Co-localisation was quantified as the
by washing with TBS (3 times for 5 min each). Sections number of co-expressing cells standardised to the total
were mounted and fluorescence preserved using ProLong number of BrdU cells imaged. CD31 DAPI images were
+
+
Gold reagent (11539306, Fisher Scientific, UK) and stored obtained from within the SC and striatal regions at 40×
+
at 4°C until imaging. For NeuN BrdU staining, acid magnification, within both contralateral and ipsilateral
antigen retrieval was performed. Sections were washed in hemispheres. For each region, three images/hemisphere/
TBS (3 times for 5 min each), incubated at 37°C in pre- slice were obtained across three slices per animal. Total
warmed two normal (2N) solutions of hydrochloric acid CD31 staining per region per animal was quantified
+
(30 min), and then neutralised using 0.1 M borate buffer following background subtraction. All image analysis
(pH 8.5; 10 min). Sections were washed with TBS (3 times was performed using FIJI ImageJ (v1.54f; NIH, United
for 5 min each), blocked for an hour and washed again, States).
as above. Before primary antibody application, residual
endogenous immunoglobulin G was blocked for 2 h with 2.12. Nissl staining
anti-rat immunoglobulin G antibody and blocking buffer, To assess delayed infarct volume, whole cortex Nissl
and sections were washed in TBS (3 times for 5 min each). staining was performed using cresyl violet acetate (CV;
Subsequently, the sections were incubated in primary C5042, Fisher, UK), using a regressive staining protocol.
antibody solution overnight at 4°C. The secondary Briefly, mounted slides were submerged in 0.1% CV
antibody staining was performed according to the steps solution for 15 min. Staining was differentiated using
above. acidified alcohol (70% industrial methylated spirit + 0.25%
Neuronal nuclei DAPI immunofluorescent images acetic acid), and slices were dehydrated using increasing
+
were acquired using fluorescence microscopy (20× air concentrations of alcohol solution. Slides were sealed using
objective lens with 0.75 numerical aperture; Zeiss LSM distrene plasticiser xylene (HX86532379, Merck, UK), air-

Volume 11 Issue 4 (2025) 79 doi: 10.36922/jctr.25.00018