CD16 Antibody - #DF7007

Fig. 5M-IHC and IHC staining of the anti-SIRPα antibody treatment model. A Representative M-IHC results of the OH2 combined anti-SIRPα antibody group, OH2 combined isotype, OH2 group, and control group with larger tumour volume sat the initial treatment. CD8, green, CD16, sky blue, F4/80, purple, CD86, orange, and CD206, red. Scale bar, 100μm. B Quantitative results of NK cell (CD16) staining in the OH2 combined anti-SIRPα antibody group, OH2 combined isotype, OH2 group, and control group. n=3 samples/group. C Quantitative result of M2 macrophage (F4/80+CD206+) staining in the OH2 combined anti-SIRPα antibody group, OH2 combined isotype, OH2 group, and control group. n=3 samples/group. D Quantitative results of M1 macrophage (F4/80+CD86+) staining in the OH2 combined anti-SIRPα antibody group, OH2 combined isotype, OH2 group, and control group. n=3 samples/group. E Quantitative results of CD8 T cell staining in the OH2 combined anti-SIRPα antibody group, OH2 combined Isotype, OH2 group, and control group. n=3 samples/group. F Representative IHC staining for CD8, CD16, F4/80, CD86, and CD206 of the OH2 combined anti-SIRPα antibody group, OH2 combined isotype group, OH2 group, and control group with larger tumour volumes at the initial treatment. Original magnification, ×200. n=5 samples/group. G Quantitative results of CD8, CD16, F4/80, CD86 and CD206 staining in the OH2 combined anti-SIRPα antibody group, OH2 combined isotype, OH2 group and control group. n=5 samples/group. Statistical analysis was performed using ANOVA with multiple comparisons. ns, no significant differences, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001 (proportion represented the percentage of the cell to the total number of cells)

Fig. 4 Effect of loganin on phenotypic transformation of macrophages. (A) The expressions of iNOS and CD16/32 in RAW264.7 cells were quantified. (B) The expressions of Arg-1 and CD206 in RAW264.7 cells were quantified. (C) The expression of iNOS in the kidney of DN mice was quantified. (D) The expression of CD206 in the kidney of DN mice was quantified. Representative results were obtained from at least three independent experiments. Data were expressed as mean ± SD. #P<0.05 and ##P<0.01 versus the normal group or the control group, *P<0.05 and **P<0.01 versus the model group or the AGEs group. The experiment was repeated three times.

Figure 4 PARP14 deficiency exacerbates the shift of M2-polarized microglia to M1-polarized microglia in mice 7 days post-SCI. (A) Representative images and quantitative analysis showing iNOS+/Iba1+ immunofluorescence staining. Lv-shPARP14 injection enhanced the SCI-induced increase in iNOS expression (pro-inflammatory phenotype). White arrows indicate iNOS+ (green, FITC-labeled)/Iba1+ (red, Cy3-labeled, microglia marker) cells. (B) Representative images and quantitative analysis showing Arg-1+/Iba1+ immunofluorescence staining. Lv-shPARP14 injection reversed the SCI-induced increase in Arg-1 expression (anti-inflammatory phenotype). White arrows indicate Arg-1+ (green, FITC-labeled)/Iba1+ (red, Cy3-labeled, microglia marker) cells. Scale bars: 50 µm in A and B. (C) Relative protein expression of CD16 and CD206 in each group. Lv-shPARP14 injection enhanced the SCI-induced increase in CD16 (M1-type marker) expression and decreased the SCI-induced increase in CD206 (M2-type marker) expression. (D) The concentration of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and anti-inflammatory cytokines (IL-10, TGF-β1, and IL-4) in each group was measured by enzyme-linked immunosorbent assay. Lv-shPARP14 injection increased the concentration of pro-inflammatory cytokines but decreased anti-inflammatory cytokine accumulation. Values are shown as mean ± SD (n = 6). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). Images were taken of the gray matter ventral horn at the injury site. Spinal cord tissues from the injury site were used for western blot and enzyme-linked immunosorbent assay detection. Arg-1: Arginase-1; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate; Iba1: ionized calcium-binding adaptor molecule 1; IL: interleukin; iNOS: inducible nitric oxide synthase; PARP14: poly(ADP-ribose)polymerase, member 14; SCI: spinal cord injury; TGF-β1: transforming growth factor-beta1; TNF-α: tumor necrosis factor alpha.

Figure 1 Exogenous BMP4-induced allodynia, microglial activation and polarization. (A) PWT in rats after intrathecal BMP4 application showed a significant effect on time: F (4.143, 66.29) = 16.09, P<0.01, left; F (3.960, 63.35) = 12.67, P<0.01, right, intrathecal application: F (1, 16) = 161.3, P<0.01, left; F (1, 16) = 160.5, P<0.01, right and interaction: F (7, 112) = 11.76, P<0.01, left; F (7, 112) = 5.653, P<0.01, right. Compared with the Sham group, rats in the BMP4 group developed a significant decrease in bilateral PWT for the whole first week, P<0.01, n=9 at each time point for both groups. (B) Representative Western blotting showed a sustained increase of CD11b (P<0.01) and CD16 (P<0.01) expressions for the whole 1st week in the BMP4 group compared with the Sham group. Meanwhile, ARG-1 levels increased at day 1 (P<0.01) and day 4 (P<0.01), then fell below the levels of the Sham group (P<0.01). n=3 for each column. (C and D) Double-immunofluorescence further detected that compared with the Sham group (P7), CD11b expression was elevated in the dorsal horn of spinal cord after BMP4 treatment. Moreover, expressions of CD16 and ARG-1 showed similar pattern with Western blotting results, which were both mainly accumulated with CD11b+ cells. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test (A) and one-way ANOVA, followed by Sidak’s multiple comparisons test (B–D) were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Figure 1 Exogenous BMP4-induced allodynia, microglial activation and polarization. (A) PWT in rats after intrathecal BMP4 application showed a significant effect on time: F (4.143, 66.29) = 16.09, P<0.01, left; F (3.960, 63.35) = 12.67, P<0.01, right, intrathecal application: F (1, 16) = 161.3, P<0.01, left; F (1, 16) = 160.5, P<0.01, right and interaction: F (7, 112) = 11.76, P<0.01, left; F (7, 112) = 5.653, P<0.01, right. Compared with the Sham group, rats in the BMP4 group developed a significant decrease in bilateral PWT for the whole first week, P<0.01, n=9 at each time point for both groups. (B) Representative Western blotting showed a sustained increase of CD11b (P<0.01) and CD16 (P<0.01) expressions for the whole 1st week in the BMP4 group compared with the Sham group. Meanwhile, ARG-1 levels increased at day 1 (P<0.01) and day 4 (P<0.01), then fell below the levels of the Sham group (P<0.01). n=3 for each column. (C and D) Double-immunofluorescence further detected that compared with the Sham group (P7), CD11b expression was elevated in the dorsal horn of spinal cord after BMP4 treatment. Moreover, expressions of CD16 and ARG-1 showed similar pattern with Western blotting results, which were both mainly accumulated with CD11b+ cells. n=3 for each column. Two-way ANOVA, followed by a Bonferroni test (A) and one-way ANOVA, followed by Sidak’s multiple comparisons test (B–D) were performed to analyze the statistical differences. *Represented P<0.05 and **Represented P<0.01 compared with the Sham group.

Figure 6 Immunofluorescence double staining of inflammatory marker CD16 and anti-inflammatory marker CD206 on microglia in the substantia nigra of mice. (A) Staining of Iba-1 (green) and CD16 (classical microglia marker, red) in the SNpc and (B) quantification of the percentage of CD16+/Iba‐1+ cell. (C) Double staining of Iba-1 (microglia marker, green) and CD206 (alternative microglia marker, red) in the SNpc for immunofluorescence pictures and (D) quantification of the percentage of CD206+/Iba‐1+ cell. Scale bar is 20 µm. Data represent the means ± SEM; Statistics one-way ANOVA; ##P < 0.01, ###P < 0.001 vs Control group; *P < 0.05, vs MPTP group; n = 3.