Fig 1: Evaluation of the regenerated nerves morphology and immunofluorescence analysis. a Cross section from the distal segments of regenerated nerves were stained by HE stain. The blue arrow indicates vessels. b Cross section from the distal segments of regenerated nerves were immunostained 8 weeks after the implantation. S100 indicated the regenerated SCs and NF200 indicated the regenerated axons. Scale bar = 100 µm. c Representative western blotting of S100 and NF200 protein expressing in regenerated nerve tissues. d Statistical analysis of S100 protein expression in regenerated nerve tissues. e Statistical analysis of NF200 protein expression in regenerated nerve tissues. Data are presented as mean ± SEM, n = 6 rats per group, *P < 0.05 by ANOVA with post hoc Bonferroni correction
Fig 2: ST2 mediates the IL-33 response on IA. A, Detection of ST2 transcripts in mouse DRGs. Neither the reverse-transcription negative control (without reverse transcriptase, -RT) nor nontemplate negative control (-H2O) showed a signal. B, Immunoblot analysis of ST2 protein abundance in mouse DRGs. Blots are representative of three independent experiments with ß-tubulin serving as a loading control. C, Colabeling (white arrows) of ST2 and NeuN, GS, CGRP, IB4 and NF200 in mouse DRG sections. Pre-incubation of ST2 antibody with excessive ST2 blocking peptide served as the specificity control of ST2 antibody. Scale bar, 50 µm. D, Time course of IA changes (left) and bar graph (right) demonstrating that pretreating DRG neurons with an ST2 neutralizing antibody (ST2 Ab, 2 µg/mL) prevented the IL-33-induced IA decrease (n = 9 cells). The application of 2 µg/mL ST2 Ab alone did not affect IA (n = 7 cells). Arabic numerals indicate the points utilized for example current traces. **p < 0.01 vs. IL-33 without ST2 Ab, paired t test. E, Immunoblot analysis of ST2 protein abundance in the control siRNA (NC-siRNA) and ST2 siRNA-treated (ST2-siRNA) groups. Blots are representative of three independent experiments with ß-tubulin serving as a loading control. **p < 0.01 vs. NC-siRNA, unpaired t test. F, Bar graph indicating that treatment with ST2-siRNA (n = 12 cells), but not NC-siRNA (n = 11 cells), abrogated the 50 ng/mL IL-33-induced IA decrease. *p < 0.05 vs. control + NC-siRNA, one-way ANOVA with a Bonferroni post hoc test.
Fig 3: Histological evaluation and immunohistostaining of spinal sacral nerve at 1 w and 4 w. (a#1-a#3 and b#1-b#3) H&E of nerve in two groups. (c#1-c#3 and d#1-d#3) S100 and NF200 immunohistostaining of spinal sacral nerve in sham (20 ×) and crush group (20 ×). (e and f) Mean density of NF200 and mean density of S100 in two groups. Data was represented as mean ± SEM. Significance levels were set at *P < 0.05.
Fig 4: TAAR1 mediates the tyramine-induced reduction in IA. a Western blot analysis of TAAR1 and TAAR4 protein expression in mouse TGs. ß-actin was used as an internal loading control. The immunoblots are representative of the results of at least three independent experiments. b Colocalization of TAAR1 (red) with NeuN, GS, IB4, CGRP or NF200 (green) in the TG (arrows). Scale bar = 50 µm. c and d The time course curve (c) and the bar chart (d) showing that pretreatment of TG neurons with EPPTB (3 µM) prevented the 0.1 µM tyramine-induced IA decrease (n = 10 cells). EPPTB at 3 µM alone did not affect IA (n = 7 cells). Insets in panel c indicate the representative traces. The letters indicate the points used for sample traces. **p < 0.01 vs. tyramine, unpaired t test. e Summary of results indicating quantified IA current density under control conditions, during exposure to RO5263397, and during washout (n = 9 cells). *p < 0.05 vs. control, paired t test. f Western blot analysis of TAAR1 protein expression in the NC-siRNA or TAAR1 siRNA-treated (TAAR1-siRNA) groups. The immunoblots are representative of the results of at least three independent experiments. **p < 0.01 vs. NC-siRNA, unpaired t test. g Representative traces (left panel) and bar chart (right panel) showing that treatment with TAAR1-siRNA abrogated the 0.1 µM tyramine-induced IA response (n = 12 cells). *p < 0.05 vs. control + NC-siRNA, unpaired t test
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