Fig 1: (See previous page). BMSCs expressing glial cell characteristic protein promoted gastrointestinal nerve regeneration in denervated mice.(A) BMSCs expressed glial markers (GFAP) after pre-induction. (B) Representative immunoblot bands and histogram of relative expression of nanog/GFAP/GDNF (normalized to GAPDH) in BMSCs after pre-conditioned for 10 days. (C) Distribution of GFP (green)/tdTomato (red)-labeled BMSCs in mouse stomach was observed by laser confocal immunofluorescence 1/3/28 days after transplantation. (D) Representative immunofluorescence images in gastric myenteric plexus of GFAP/ß-tubulin (green) and tdTomato-labeled BMSCs (red) at 28 days after transplantation. GFAP+ (green) cells in the myenteric ganglia emerge that express tdTomato (red) and fluoresce yellow (marked here by white arrow). (E) Representative immunofluorescence confocal laser images in transverse sections of gastric tissue sections of GFAP/ß-tubulin (green) in each group (Control/Sham/BAC/BAC+BMSCs), the nuclei (blue). (F) Representative immunoblot bands and histogram of relative expression for GDNF/GFAP/HuC/D/ß-tubulin in the gastric tissues (normalized to GAPDH). S, subserosa; LM, longitudinal muscle; CM, circular muscularis; SM, submucosa; M, mucosal layer; Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; BAC+BMSCs, BAC treated mice transplanted with BMSCs. These results are representative of at least 3 times independent experiments. Results were expressed as mean ± standard deviation. **P < .01, ***P < .001, ****P < .0001.
Fig 2: BAC (0.05%) effectively ablated the myenteric plexus in gastric tissue and remained at least 31 days.(A) Representative immunofluorescence confocal laser images in transverse sections of gastric tissue sections of PGP 9.5 (red) in the control group (upper panel) and 0.05% BAC treated group (lower panel); the a-SMA (green); the nuclei (blue). (B–D) Representative immunofluorescence confocal laser images in gastric myenteric plexus of PGP 9.5 (red), HuC/D (red), and GFAP (red) in each group; the nuclei (blue). (E and F) Representative immunoblot bands and histogram of relative expression for PGP 9.5/HuC/D/GFAP in each group (normalized to GAPDH). Control, the control C57 mice; BAC, benzalkonium chloride treated C57 mice. Results were expressed as mean ± standard deviation. ****P < .0001, n = 4–5.
Fig 3: Proportion of Nestin+/Ngfr+cells differentiated into neurons was significantly higher than that of Nestin+cells.(A) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (green) and tdTomato-labeled Nestin+ cells (red) in BAC+BMSCs+TAM (Nestin-creERT2: tdTomato mice), the nuclei (blue). Arrows indicate double-positive cells. (B) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/ß-tubulin/HuC/D/nNOS (red) and GFP-labeled Nestin+/Ngfr+ cells (green) in BAC+BMSCs+TAM (Nestin-creert2 X Ngfr-Dreert2: DTRGFP mice), the nuclei (blue). Arrows indicate double-positive cells. (C and D) Proportion of Nestin+ (red)/Nestin+Ngfr+ (blue) cells differentiated into neurons and glial cells in Control+TAM group and BAC+BMSCs+TAM group. Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; TAM, tamoxifen. These results are representative of independent experiments repeated at least 3 times. Results were expressed as mean ± standard deviation. *P < .05, **P < 0.01, NS, no significance.
Fig 4: Regenerated neurons derived from Nestin+cells in denervated double-transgenic mice (Nestin-creERT2: tdTomato).(A–D) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/β-tubulin/ HuC/D/nNOS (green) and tdTomato-labeled Nestin+ cells (red) in Control, Control+TAM, Sham+TAM, and BAC+TAM groups, the nuclei (blue). Arrows indicate double-positive cells. Nestin+ cells are stained with td-Tomato (red) and express GFAP/β-tubulin/ HuC/D/nNOS (green) after TAM induction and show co-localization, providing evidence that Nestin+ cells can be differentiated into neurons and glial. (E and F) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/β-tubulin/ HuC/D/nNOS (gray), tdTomato-labeled Nestin+ cells (red), and GFP-labeled BMSCs (green) in Sham+BMSCs+TAM and BAC+BMSCs+TAM groups, the nuclei (blue). Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; BAC+BMSCs, BAC treated mice transplanted with BMSCs; TAM, tamoxifen.
Fig 5: Regenerated neurons derived from Nestin+/Ngfr+cells in denervated triple-transgenic mice (Nestin-creERT2 × Ngfr-DreERT2: DTRGFP).(A–D) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/β-tubulin/HuC/D/nNOS (red) and GFP-labeled ENPCs (green) in Control, Control+TAM, Sham+TAM, and BAC+TAM, the nuclei (blue). Arrows indicate double-positive cells. Nestin+/Ngfr+ cells are stained with GFP (green) and express GFAP/β-tubulin/ HuC/D/nNOS (red) after TAM induction and show co-localization providing evidence that Nestin+/Ngfr+ cells can be differentiated into neurons and glial. (E and F) Representative immunofluorescence confocal laser images in freshly intermuscular plexus tissues of gastric of GFAP/β-tubulin/HuC/D/nNOS (gray), GFP-labeled ENPCs (green), and tdTomato-labeled BMSCs (red) in Sham+BMSCs+TAM and BAC+BMSCs+TAM groups, the nuclei (blue). Control, the control C57 mice; Sham, sham operation group; BAC, benzalkonium chloride treated group; BAC+BMSCs, BAC treated mice transplanted with BMSCs; TAM, tamoxifen.
Supplier Page from Abcam for Anti-HuD + HuC antibody [EPR19098]