Fig 1: Presence of GGT1 or GGT7 in mouse and human olfactory epithelium(A) Single-cell RNA-seq t-SNE plots and violin plots with overlaid log normalized expression levels of the Ggt1 and Ggt7 genes in the mouse nasal epithelium.(B) Anti-GGT7 staining of a coronal section through a mouse nasal septum corresponding to the boxed area labeled “B” in Figure 1A mainly labels the apical surface of sustentacular cells (arrows), glands (arrowheads), and duct cells (open arrowheads) in OE. Scale bar = 50 µm.(C) Immunofluorescent staining of the boundary between OE and RE corresponding to the boxed area labeled “C” in Figure 1A with antibodies against TUJ1 to label OSNs and CK19 to label respiratory cells shows anti-GGT7 labeling confined to sustentacular cells (arrows) in OE. Scale bar = 25 µm.(D) Relative mRNA expression of Ggt7 and Ggt1 in mouse OE and RE. The data are shown as the mean ± SEM (n = 4). *p < 0.05.(E) Immunofluorescent staining of OE from an OMP-tTA; TetO-DTA transgenic mouse from the boxed areas labeled “G-I” in Figure 1F illustrates the selective staining of anti-GGT7 for neuronal OE. The neuronal OE contains TUJ1(+) OSNs as well as GGT7(+) sustentacular cells (arrows) and glands (arrowheads). The aneuronal OE lacks staining with TUJ1, TUBB4, and GGT7. Areas of respiratory metaplasia contain TUBB4(+) respiratory cells but lack TUJ1 and GGT7 staining. Scale bar = 25 µm.(F) Sections of human autopsy samples corresponding with boxed areas labeled “K-M” in Figure 1J are labeled with anti-GGT7. The neuronal OE contains TUJ1(+) OSNs as well as GGT7(+) sustentacular cells (arrows). The aneuronal OE lacks TUJ1, TUBB4, and GGT7 staining. Respiratory metaplastic epitlelium consists of TUBB4 positive cells but lacks TUJ1 and GGT7 staining. Scale bar = 25 µm.(G) Single-cell RNA-seq t-SNE and violin plots with expression levels of the GGT7 gene in the human nasal epithelium.
Fig 2: a Viability of 16HBE cells exposed to TNF-a with or without LHQK treatment. Data are presented as Mean ± SD (n = 3 in each group). *P = 0.05 vs TNF-a group. b Effects of LHQK on MUC5AC expression induced by TNF-a in 16HBE cells. Data are presented as Mean ± SD (n = 5 in each group). *P = 0.05, **P = 0.01 vs TNF-a group. c Immunofluorescence labelling of airway cilia ß-IV tubulin in rat bronchial primary epithelial cells (RTE). Cells were stained with anti-ß-IV tubulin (ab179509, Abcam, USA) followed by Alexa 555-conjugated secondary antibody. Nuclei were counterstained with DAPI. Samples were imaged using a ZEISS LSM710. Scale bar, 20 µm
Fig 3: Vital imaging of nasal mucosa after application of coumarin demonstrates labeling of OE(A) Coumarin is catalyzed to 7OH-coumarin by CYP2A6.(B) Whole mount staining of a normal mouse nasal cavity using antibodies against PGP 9.5 to label OSNs, and TUBB4 to label respiratory cells reveal the demarcation between OE and RE on the lateral wall with turbinates and septum.(C) Fluorescent imaging of whole mounts loaded with 10 mM coumarin captured before and 5, 15, and 30 min after probe application reveal signal in areas comparable to OE.(D) Relative fluorescence intensity of OE vs. RE measured every 5 min after application of coumarin. The data are shown as the mean ± SEM (n = 5). Statistical differences were calculated in initial time vs 30 min and initial time vs 60 min *p < 0.05. D = dorsal, V = ventral, A = anterior, p = posterior, OB = olfactory bulb, scale bars = 2 mm.
Fig 4: Anti-CYP2A6 staining is identified in mouse and human OE(A) Coronal section through the nasal cavity of a mouse stained with H&E. Scale bar = 1 mm.(B) Anti-CYP2A6 staining of nasal septum OE; the area illustrated corresponds to the boxed area in Figure 1A. CYP2A6 staining is mainly located in sustentacular cells especially at the apical surface (arrows) and glands (arrowheads). Scale bar = 25 µm.(C) IHC with anti-CYP2A6 staining recognizes the boundary between OE and RE. The area illustrated corresponds to the boxed area in Figure 1A. Antibodies against PGP 9.5 label olfactory sensory neurons (OSNs) of OE, while CK19 antibodies label columnar ciliated cells of the RE. CYP2A6 staining is found only in the sustentacular cells in OE (arrows) and is absent in RE. Scale bar = 25 µm.(D) Relative mRNA expression of Cyp2a5 in mouse. The data are shown as the mean ± SEM (n = 4). *p < 0.05.(E) Single-cell RNA-seq t-SNE and violin plots with expression levels of the Cyp2a5 gene in the mouse nasal epithelium.(F) IHC of the nasal septum from an OMP-tTA; TetO-DTA transgenic mouse model. A mosaic image of a section labeled with anti-TUJ1 identifies OSNs (box G), and labeled with TUBB4 to identifies RE (box I). Areas that have become aneuronal are absent of labeling with both antibodies (box H). Scale bar = 100 µm.(G) The neurogenic OE contains PGP9.5 (+) OSNs as well as CYP2A6 (+) sustentacular cells (arrow) and glands (arrowhead).(H) The aneuronal OE lacks PGP9.5/TUBB4/CYP2A6 staining.(I) Respiratory metaplastic epithelium contains TUBB4 (+) columnar respiratory cells but lacks PGP9.5/CYP2A6 staining. Scale bars = 25 µm.(J) A mosaic image of a human section through the OE obtained at autopsy (74-year-old male) includes neuronal OE (TUJ1+/TUBB4-, box K), RE (TUJ1-/TUBB4+, box M), and patches of aneuronal OE (TUJ1-/TUBB4-, box L). D = dorsal, V = ventral. Scale bar = 250 µm.(K) Neuronal OE contains PGP9.5(+) OSNs as well as CYP2A6(+) sustentacular cells with high labeling at the apical surface (arrows).(L) Aneuronal OE lacks both PGP9.5 and TUBB4 staining.(M) RE contains TUBB4(+) columnar epithelial cells but lacks both PGP9.5 and CYP2A6 labeling. Scale bars = 25 µm.
Fig 5: Vital imaging can define areas of neuronal OE regeneration after lesioning(A) Whole mount staining of the nasal cavity from a methimazole/dichlobenil lesioned mouse using PGP 9.5/TUBB4 identifies areas on both the lateral wall and septum where respiratory metaplasia (arrowheads) in previous neuronal OE. D = dorsal, V = ventral, A = anterior, p = posterior, OB = olfactory bulb. Scale bars = 2 mm.(B) A coronal section of the nasal cavity from a methimazole/dichlobenil lesioned mouse demonstrates respiratory metaplasia in the dorsal area of OE (arrows). The arrowhead indicates intracavitary excrescences from the injured OE. Scale bar = 1 mm.(C) Regenerated ventral OE from the boxed area labeled “C” in Figure 5B contains a thick layer of TUJ1 labeled OSNs without TUBB4+ cells. Scale bar = 25 µm.(D) A dorsal region of OE from the boxed area labeled “D” in Figure 5B is without neurons and is labeled for respiratory cells with TUBB4 antibodies. Scale bar = 25 µm.(E) A cartoon illustrates wholemount staining in a methimazole/dichlobenil-injected mouse with respiratory metaplasia in the dorsal recess of the nasal cavity.(F) Fluorescent imaging of whole mounts incubated with 10 mM coumarin captured before and 30 min after probe application reveals signal in the ventral area of OE but not in dorsal areas of what was formerly OE (arrowheads).(G) Relative fluorescence intensity of dorsal vs. ventral mucosa measured every 5 min after application of coumarin. The data are shown as the mean ± SEM (n = 5).(H) Fluorescent imaging of whole mounts incubated with 10 µM Glu-HMRG captured before and 30 min after probe application again reveals signal in the ventral area of OE but not the dorsal area (arrowheads).(I) Relative fluorescence intensity of dorsal vs. ventral mucosa measured every 5 min after application of gGlu-HMRG. The data are shown as the mean ± SEM (n = 5). D = dorsal, V = ventral, A = anterior, p = posterior, OB = olfactory bulb, scale bars = 2 mm.
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