Journal: BMC Genomics

Article Title: Lipid metabolism-related genes are involved in the occurrence of asthma and regulate the immune microenvironment

doi: 10.1186/s12864-023-09795-3

Figure Lengend Snippet: GSEA enrichment analysis. A - C GSEA enrichment plots of hub genes ( ASAH1 , ACER3 and SGPP1 ). GSEA: gene set enrichment analysis

Article Snippet: Samples were incubated with anti-rabbit ASAH1 antibody (1:80, Proteintech, Chicago, United States), anti-rabbit ACER3 (1:200, Proteintech, Chicago, United States), and anti-rabbit -SGPP1 antibody (1:200, Affinity, Ancaster, Canada).

Techniques:

Journal: BMC Genomics

Article Title: Lipid metabolism-related genes are involved in the occurrence of asthma and regulate the immune microenvironment

doi: 10.1186/s12864-023-09795-3

Figure Lengend Snippet: Correlation analysis between hub genes and 22 immune cells. A - C The lollipop diagram showing the relationship of hub genes ( ASAH1 , ACER3 and SGPP1 ) and immune cells

Article Snippet: Samples were incubated with anti-rabbit ASAH1 antibody (1:80, Proteintech, Chicago, United States), anti-rabbit ACER3 (1:200, Proteintech, Chicago, United States), and anti-rabbit -SGPP1 antibody (1:200, Affinity, Ancaster, Canada).

Techniques:

Journal: BMC Genomics

Article Title: Lipid metabolism-related genes are involved in the occurrence of asthma and regulate the immune microenvironment

doi: 10.1186/s12864-023-09795-3

Figure Lengend Snippet: Correlation of ASAH1 with differentially-expressed immune cells. A , B Association between ASAH1 and CD8 + T cell and activated NK cells

Article Snippet: Samples were incubated with anti-rabbit ASAH1 antibody (1:80, Proteintech, Chicago, United States), anti-rabbit ACER3 (1:200, Proteintech, Chicago, United States), and anti-rabbit -SGPP1 antibody (1:200, Affinity, Ancaster, Canada).

Techniques:

Journal: BMC Genomics

Article Title: Lipid metabolism-related genes are involved in the occurrence of asthma and regulate the immune microenvironment

doi: 10.1186/s12864-023-09795-3

Figure Lengend Snippet: Correlation of ASAH1 with differentially-expressed immune cells. A , B Association between ASAH1 and monocytes and M0 macrophages

Article Snippet: Samples were incubated with anti-rabbit ASAH1 antibody (1:80, Proteintech, Chicago, United States), anti-rabbit ACER3 (1:200, Proteintech, Chicago, United States), and anti-rabbit -SGPP1 antibody (1:200, Affinity, Ancaster, Canada).

Techniques:

Journal: BMC Genomics

Article Title: Lipid metabolism-related genes are involved in the occurrence of asthma and regulate the immune microenvironment

doi: 10.1186/s12864-023-09795-3

Figure Lengend Snippet: The result of validation by immunofluorescent staining and RT-qPCR in mouse models ( A – D ) immunofluorescent staining images of lung tissues and quantification of fluorescence intensity, Nucleus (blue), ASAH1/ACER3/SGPP1 (red), scale bar, 500 um, ( n = 4 per group) ( E – G ) The expression of ASAH1 , ACER3 , and SGPP1 verified by RT- qPCR in the OVA model ( n = 4 per per)

Article Snippet: Samples were incubated with anti-rabbit ASAH1 antibody (1:80, Proteintech, Chicago, United States), anti-rabbit ACER3 (1:200, Proteintech, Chicago, United States), and anti-rabbit -SGPP1 antibody (1:200, Affinity, Ancaster, Canada).

Techniques: Biomarker Discovery, Staining, Quantitative RT-PCR, Fluorescence, Expressing

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Principle of unbiased correlation using intrinsic landmarks. IF channels are locked and hidden behind nuclear DAPI signal, which is used as only visible fluorescent channel, applied as landmark channel (blue). DAPI signal and SEM nucleus texture are used to achieve unbiased correlation of IF and SEM information. After optimization of the correlation, the channels of interest are made visible, overlaid and regions of interest selected. No further changes of image locations were applied afterwards. IF signal specificity. ( B ) Four consecutive tissue sections with nuclear DAPI (blue), DP-1 (yellow), and PGP9.5 (magenta) labeling showing traceable persistent labeling. Arrowhead indicates PGP9.5 positive nerve fiber and numbers show position of respective nuclei. Nucleus number 2 is ceasing within the z-stack. Scale bar 5 µm. ( C ) Application of secondary antibodies Al488-anti mouse (green), SeTau-647-anti rabbit (yellow), and Cy3-anti guinea pig (red) alone showed no fluorescent signal. DAPI signal (blue) for orientation. Scale bar 10 µm. Abbreviations: Al488, Alexa Fluor 488; Cy3, cyanine 3; DAPI, 4',6-diamidino-2-phenylindole; DP-1, desmoplakin 1; IF, immunofluorescence, PGP9.5, protein product 9.5 SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Tracking of Cx43 plaques in epidermal layers. First panel illustrates SEM overview of epidermal layers corresponding to five consecutive sections of IF images showing Cx43 signal (red) and nuclei (blue). Asterisks mark examples of traced Cx43 plaques. ( B ) Cx43 plaques at keratinocyte-nerve fiber close contact sites. Nerve fibers processing between keratinocytes in skin samples of two male subjects (M1, M2) and one female subject (F1). Each row represents four consecutive sections of 100 nm thickness. First tile shows keratinocyte cell bodies (orange), nuclei (blue), and nerve fibers (magenta) in pseudo color with Cx43 signal (red). Correlated PGP9.5 labeling (magenta) locates at nerve fibers and Cx43 labeling (yellow) indicates Cx43 plaques. Insets show magnification of contact area. Scale bars: 5 µm ( A ), 1 µm ( B ), magnified insets: 500 nm. Abbreviations: Cx43, connexin 43; IF, Immunofluorescence; PGP9.5, protein gene product-9.5; SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Overview of skin section prior to expansion with PGP9.5-labeled IENF (magenta), Cx43 (yellow), and nuclear DAPI (blue); dotted line illustrates epidermis-dermis border and arrow indicates IENF. White rectangle marks inset enlarged in B. ( B ) Enlarged area prior to expansion and ( C ) matched area post-expansion at same magnification with addition of actin marker phalloidin (grey); white rectangles mark inset area of D and E. ( D ) Enlarged IENF area at ×20 magnification and ( E ) at ×63 magnification with arrowheads indicating Cx43 plaque at IENF. Inset in E marks enlarged area in E`-E```. ( E` ) shows z-plane prior to E, ( E`` ) same z-plane as E, and E``` z-plane step after E. White scale bars indicate pre-expansion state, yellow scale bars are corrected for expansion factor. Scale bars: 50 µm ( A ), 5 µm ( B, C ), 2 µm ( D, E ), 500 nm ( E`-E``` ). Z-step size of 1.2 µm translates to approximate 274 nm in expanded gel. Abbreviations: Cx43, connexin 43; IENF, intraepidermal nerve fiber; PGP9.5, protein gene product-9.5.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Marker, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Workflow for ensheathment and Cx43 quantification (see also ). ( B ) Magnitude of skin tissue expansion across investigated sections via comparison of epidermal nuclei. Median nucleus size between samples pre-ExM (8.73±0.54 µm) and post ExM (32.46±3.54 µm) denotes a median ExM factor of 3.83±0.36. ( C ) Extracted fiber ensheathment ratio (PGP9.5-positive px without WGA pixel co-localization divided by total PGP9.5 px count) in HC versus SFN patients. ( C ) Extracted fiber ratio (total PGP9.5 px versus PGP9.5 non WGA overlap px) in HC versus SFN patients (n=13 HC and 10 HC ROIs from n=5 HC and n=4 SFN samples; p=0.41; shown as box & whiskers plot with minimum and maximum). ( D ) Cx43 contact ratio (PGP9.5 px with Cx43-WGA double positive px divided by total PGP9.5 px) in HC versus SFN (n=13 HC and 10 SFN ROIs from n=5 HC and n=4 SFN samples; p=0.34; shown as box & whiskers plot with minimum and maximum). Expansion factor corrected scale bars (yellow) of exemplary segmentation: 5 µm. See also . Abbreviations: HC, healthy control; Cx43, connexin 43; NCU, neuro-cutaneous unit; PGP9.5, protein gene product 9.5; px, pixel; SFN, small fiber neuropathy; WGA, wheat germ agglutinin. Figure 5—source data 1. Descriptive data and values for nucleus based calculation of expansion factors. Figure 5—source data 2. Statistical details and input values for .

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Comparison, Control, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43 positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta) and nuclear DAPI (blue). ( B ) Inset of ensheathment area from A. ( C ) Single plane lattice SIM image and respective inset area with 3D visualization of z-stack (2.925 µm depth, 0.196 µm steps) showing nucleus, Cx43, and neurite signal ( C` ) and including WGA ( C`` ). ( D ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43-positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta), and nuclei (blue). Arrowheads indicate Cx43 - neurite contact areas. ( E ) Single plane lattice SIM image and respective inset area with 3D visualization, numbers represent single Cx43 plaques ( F ) of z-stack (2.925 µm depth, 0.196 µm steps). ( F`-F`` `) detailed neurite - Cx43 contact areas. Co-culture kept in keratinocyte medium. Scale bars: 20 µm ( A, D ), 5 µm ( C-C``, E, F`-F```` ), 1 µm ( F ). Abbreviations: Ctx, cholera toxin subunit B; Cx43, connexin 43; DAPI, 4',6-diamidino-2-phenylindole; SIM, structured illumination microscopy; WGA, wheat germ agglutinin.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Confocal, Membrane, Labeling, Co-culture Assay, Microscopy, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: Primary and secondary antibodies and marker with specifications.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Marker

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques:

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Immunofluorescence