Journal: Gastroenterology

Article Title: Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

doi: 10.1053/j.gastro.2020.02.035

Figure Lengend Snippet: (A) Flattened Z-stack through myenteric plexus (10x objective, stitched fields, 4x5mm2). Small clustered ganglia have HuC/D+ (green) and PHOX2B+ (blue) neurons. Nerve fibers are visualized via glial S100β (red). Myenteric plexus and ganglia within plexus were outlined manually (thin yellow lines). (B) Quantitative data from 4x5mm2 images like (A). (C,D) Flattened Z-stack from colon myenteric ganglia (left (C) or right (D)) stained for HuC/D (green), S100β (red) and PHOX2B (blue) imaged with 20x objective. Table to right of each image shows quantitative data from that specific Z-stack. (E) Quantitative data from 4 right colon, 5 fields/subject; 3 left colon, 5 fields/subject; 1 left colon, 4 fields; Total neurons counted: 1953 right colon, 2603 left colon; Total glia counted: 8021 right colon, 6423 left colon. Single ganglion areas are from 24 right and 19 left colon ganglia. Cells/mm2 within these ganglia are based on 5261 neurons and 16,915 glia. (F,G,H) Colon stained with HuC/D (green), S100β (red), and PHOX2B (blue) antibodies. PHOX2B+ nuclei include all myenteric neurons and glia within myenteric ganglia (G). Glia in longitudinal (F) and circular muscle (H) were not PHOX2B immunoreactive. (A) Scale bar = 1000μm. (C,D) Scale bar=350μm. (F-H) Scale bar=50μm.

Article Snippet: Zeiss LSM 710 confocal microscope (10x and 20x Plan-Apochromat objectives, Zeiss Zen software (version 2.3 14.0.14.201)) Z-axis increments were 4μm (10x objective) or 1μm (20x objective).

Techniques: Staining

Journal: Gastroenterology

Article Title: Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

doi: 10.1053/j.gastro.2020.02.035

Figure Lengend Snippet: (A, B) Three-dimensional ganglia volumes (yellow regions) (Supplemental Videos 5 and 6 are from these regions). To right of each image are quantitative data from that specific region. (C) Cell density based on five 20x fields/subject, 8 subjects (4 left, 4 right colon)), 24 right and 19 left colon ganglia volumes, 5261 neurons and 16,915 glial cells. (D-M) Manual neuron and glia counts were obtained after HuC/D (blue), S100β (green) and PHOX2B (red) staining. (D,I) Flattened Z-stacks. (E-H,J-M) Single slices and channels from Z-stack. (N,O) To estimate cell density over large regions we multiplied density within small regions (like Figure 2C) by percentage of bowel with myenteric plexus (using images like 2A). We found little variability in cell density within ganglia, indicated by tight data clusters in individuals. Inter-individual differences primarily reflect percentage of bowel occupied by ganglia in each individual. (A,B,D,I) Scale bars=100 μm. (E,F,G,H,J,K,L,M) Use (E) scale bar=25 μm.

Article Snippet: Zeiss LSM 710 confocal microscope (10x and 20x Plan-Apochromat objectives, Zeiss Zen software (version 2.3 14.0.14.201)) Z-axis increments were 4μm (10x objective) or 1μm (20x objective).

Techniques: Staining