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. 2023 Sep 26;17:1277268. doi: 10.3389/fncel.2023.1277268

Figure 3.

Figure 3

Immunofluorescence analysis of NLRP3 inflammasome at 6-h, 24-h, or 72-h following I/R. (A) Immunofluorescence staining of NLRP3. Nuclei were stained with Hoechst. Representative images of NLRP3 (red) and Hoechst-33258 (blue) were overlapped (40X, scale bar = 25 μm). Cytoplasmic NLRP3 labeling was increased in the DMSO groups of the acute and subacute phases of I/R. By the administration of ML351, labeling and signal intensity of NLRP3 were dramatically decreased at all time points following I/R. (B) Graphical representation of the changes in cell numbers with positive NLRP3 immunoreactivity at 6, 24, and 72-h. NLRP3 positive cell numbers were increased in both the acute (6 and 24-h) and subacute (72-h) phases of I/R. The decreases in NLRP3 immunoreactivity at 6-h (***p = 0.0003), 24-h (**p = 0.0027) and 72-h (****p < 0.0001) with ML351 treatment were statistically significant (n = 3 mice/group). (C) Double immunolabeling of NLRP3 (red) and neurons (NeuN; neuronal marker) (green) showed that NLRP3 is colocalized with NeuN (white arrows) signal especially at 6-h and 24-h after I/R. At 72-h of I/R NLRP3 immunoreactivity was present both in neurons (white arrows) and non-neuronal cells (black arrows). (D) Comparison of NLRP3 and NLRP1 staining in I/R-DMSO groups normalized to naive brain tissue. NLRP3 immunostaining was significantly increased compared to NLRP1 immunoreactivity (****p < 0.0001).