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. 2023 Feb 10;24(4):3598. doi: 10.3390/ijms24043598

Figure 3.

Figure 3

Temporal expression of NETosis in the ipsilateral brain samples after HI-NT or HI-TH. Representative Western blotting images for all time points and conditions (a) for neutrophil NETosis marker Cit-H3 and β-actin as loading control, or (c) MPO marker and β-actin. (b) Quantitative analysis of Cit-H3 expression or (d) MPO expression in neutrophils isolated from the brain samples at the indicated time points after HI following NT or TH. The optical density was measured, and the ratio against the loading control β-actin was calculated. Sham (white column), HI (grey with cross-line column), HI/NT (grey column), and HI/TH (black column). Data of ten biological replicates (n = 10) per time point and condition (see Section 4). Two-way ANOVA was used for c and e while Student’s t-test was used for g with a * p < 0.05. Data are expressed as the mean ± SD. (e) Representative images of positive NETosis in the cortical area from the ipsilateral side of the brain 24 h post-HI. The following antibodies were used: Cit-H3 (green), neutrophils marker Ly6g (red), and nuclear marker Dapi (blue). The white arrow indicates positive Cit-H3/Ly6g NETs and the corresponding nuclear staining. Images were taken with confocal LSM900, with a 20× objective and a zoom of 3×. Scale bar = 20 µm. (f) Quantitative analyses of the cortical area showing the number of positive Cit-H3 cells in the NT group (grey column) compared to the TH group (black column) (24 h after HI; data from five biological replicates (n = 5)). Student’s t-test was used with a * p < 0.05. Data are expressed as the mean ± SD. (g) Representative image of double-immunostaining for NETs and the neutrophil marker MPO in the cortical area from the ipsilateral side of the brain 24 h post-HI. The following antibodies were used: Cit-H3 (green), neutrophils marker MPO (red), and the nuclear marker Dapi (blue). The white arrow indicates positive Cit-H3 NETosis and the corresponding MPO-positive cells. Images were taken with confocal LSM900, with a 20× objective and a zoom of 2×. Scale bar = 10 µm. (h) Representative three-dimensional pictures of double immunostaining for Cit-H3 and MPO in the cortical area. Each square on top of each picture represents a dimensional plane (up—y/x, right side y/z) where it is possible to observe the co-localization between both markers. Scale bar = 20 µm. (i) Representative images of multiple immunostaining for NET marker Cit-H3 (green), neutrophil marker MPO (red), blood vessel marker Isolectin 4 (purple), and nuclear marker Dapi (blue) in the cortex area from the ipsilateral side of the brain 24 h after HI. The white arrow indicates positive Cit-H3 NETosis and the corresponding MPO-positive cell. Images were taken with confocal LSM900, with a 20× objective and a zoom of 2×. Scale bar = 10 µm.