Journal: iScience

Article Title: Single-cell atlas of the aging mouse colon

doi: 10.1016/j.isci.2022.104202

Figure Lengend Snippet:

Article Snippet: Ulex Europaeus Agglutinin I (UEA-1), Dylight® 649 coupled , Vector Labs , Cat# DL-1068-1.

Techniques: Recombinant, Lysis, Plasmid Preparation, Multiplex Assay, Staining, RNA Sequencing Assay, Sequencing, Real-time Polymerase Chain Reaction, Software, Imaging, Light Microscopy, Microscopy, Inverted Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1, PHF-tau, GFAP, and CLDN5 immunoreactivities in the CA1 area of dementia brains. (A) Representative image of location in human Control hippocampus of area ca1. Magnification: 10×. Scale bar: 25 μm. (B) Representative images of the BACE1, (C) PHF-tau, (D) GFAP, and (E) CLDN5 immunoreactivities in the CA1 area of human hippocampal tissue. Magnification: 10×. Scale bar: 50 μm. Inset: 25 μm scale bar. The values in the bar graph are expressed as a densitometric percentage of the BACE1 IR in the CA1 area. FAD, familial-type Alzheimer’s disease (presenilin 1 mutation E280A); SAD, sporadic Alzheimer’s disease; CADASIL, autosomal dominant cerebral arteriopathy with subcortical infarcts and leukoencephalopathy. The data are expressed as the means ± SEM. n = 4. ∗ p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1 association with astrocytes and vessels in dementia brains. (A) Representative protein bands from the immunoprecipitation of BACE1 and co-inmunoprecipitation of GFAP are shown. IgG was used as a negative control for immunoprecipitation. (B) Z projection of Immunofluorescence of blood vessels triple-stained with DyLight 649- tagged UEA (color-coded in blue), anti-BACE1 antibody probed with Alexa Fluor 488 (green) and reactive astrocytes marked with Alexa Fluor 594 (red) in the hippocampal CA1 area. Magnification: 60x. Scale bar: 25 μm. Insets: 5 μm scale bar in B. (C) Z projection of the deconvolved images from the 3D reconstruction of the confocal images at 0 to 10 μm away from the vessel surface. Scale bar: 25 μm. The values represent the volumes in μm 3 for the levels of BACE1 and GFAP and the colocalization of BACE1 and GFAP. The data are expressed as the means ± SEM. n = 4. * p < 0.05.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Immunoprecipitation, Negative Control, Immunofluorescence, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1 inhibition protects ECs from the damaging effects of glutamate. (A) Percentage of LDH release by ECs (bEnd.3) after 24 h of treatment. The data are presented as the mean ± SEM of n = 7. Kruskal-Wallis test. (B) Percentage of condensed nuclei for each treatment. These were quantified from 20 fields per treatment for each n ( n = 4). The data are presented as the means ± SEM. (C) Number of gaps per field from threshold image (gaps = spaces between cells indicating disruption of the monolayer) presented as the mean ± SEM of n = 4. Kruskal-Wallis test followed by Dunn’s test with the Bonferroni correction. (D) Area of gaps between cells expressed in μm 2 ; the area was quantified as the mean of the black area in threshold images from 20 fields per treatment for each n ( n = 4). ANOVA followed by Tukey’s test. (E) FI of CLDN5 per unit area was quantified from 20 cells per treatment for each n ( n = 4 performed in duplicate) and divided by unit area. f) Morphological characterization of ECs showing the nuclei stained with Hoechst (blue), the F-actin cytoskeleton visualized with Alexa Fluor 488 phalloidin (green), and the tight junction protein CLDN5 visualized with Alexa Fluor 594 (red). Magnification: 60x. Scale bar: 20 μm. The zoomed insets show the condition of the nuclei, the state of the actin cytoskeleton and the distribution of CLDN5. (G) Fluorescence profiles (right column) showing the cellular distribution of CLDN5 (red line) in relation to the nucleus (blue line) and cytoskeleton (green line) based on the representative images (left column). The black arrows indicate the tight junction peaks to show the changes in CLDN5 distribution by the treatment. The white line drawn on each cell represents a distance of 50 μm. The data are presented as the means ± SEM, ANOVA followed by Tukey’s test. DMSO = vehicle, Inh = β-secretase inhibitor IV, - or Glu refers to glutamate treatment. All experiments were performed in duplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Inhibition, Staining, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1 inhibitor reduces the glutamate-induced increases in BACE1 IR and the IL-1β inflammation marker in ECs. (A,B) Fluorescence intensities of BACE1 and IL-1β, respectively, per unit area, quantified from 20 cells per treatment for each n ( n = 4 performed in duplicate) and divided by unit area. (C) Representative images showing BACE1 (red, visualized with Alexa Fluor 594) and IL-1β (green, visualized with Alexa Fluor 488) expression in ECs (bEnd.3) under different treatments. Magnification: 60x. Scale bar: 20 μm. The data are presented as the means ± SEM, ANOVA followed by Tukey’s test. *** P < 0.001. The bEnd.3 cell cultures were pretreated on DIV 8 with β-secretase inhibitor IV (CAS 797035-11-1, Merck) at 1 μM. Twenty-four hours later, the cells were treated with glutamate at 125 μM for 20 min and were subsequently treated with the inhibitor again. On DIV 10 the cells were fixed for immunofluorescence staining.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Marker, Fluorescence, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1 inhibition reduces astrocytic reactivity and induces filopodia-like processes. (A) Astrocytic cytotoxicity expressed as the percentage of LDH release after 24 h of treatment. The data are presented as the means ± SEM of n = 8. Kruskal-Wallis test followed by Dunn’s test with Bonferroni correction. (B) FI of GFAP per unit area, quantified from 20 cells per treatment for each n ( n = 4) and divided by unit area. The data are presented as the means ± SEM. ANOVA followed by Tukey’s test. (C) FI of BACE1 per unit area, quantified from 20 cells per treatment for each n ( n = 4) and divided by unit area. ANOVA followed by Tukey’s test. (D) FI of IL-1β per unit area, quantified from 20 cells per treatment for each n ( n = 4) and divided by unit area. (E) Morphological characterization of astrocytes showing the nuclei stained with Hoechst (blue), the F-actin cytoskeleton visualized with Alexa Fluor 594 phalloidin (red), and GFAP visualized with Alexa Fluor 488 (green). Magnification: 60x. Scale bar: 20 μm. In the zoomed insets, the white arrows indicate projections of the actin cytoskeleton, and the yellow arrows indicate extracellular GFAP IR puncta. (F) Representative images showing BACE1 (red, visualized with Alexa Fluor 594) and IL-1β (green, visualized with Alexa Fluor 488) expression in primary astrocytes under different treatments. The zoomed insets show the distributions of both proteins and an apparent perinuclear colocalization in vesicles (yellow). Magnification: 60x. Scale bar: 20 μm. DMSO = vehicle, Inh = β-secretase inhibitor IV, - or Glu refers to glutamate treatment. Kruskal-Wallis test followed by Dunn’s test with Bonferroni correction. The data are presented as the means ± SEM, and the experiments were performed in duplicate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Inhibition, Staining, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1-inhibited astrocytes protect the integrity of cocultured ECs from damage caused by glutamate. (A) Percentage of LDH release by the astrocyte-endothelium coculture after different treatments. (B) Percentage of condensed nuclei for each treatment, quantified from 20 fields per treatment for each n. (C) The number of gaps per field from threshold images. (D) The area of gaps between cells expressed in μm 2 , quantified as the mean of the black area in threshold images from 20 fields per treatment for each n. (E) FI of IL-1β per unit area, quantified from 20 cells per treatment for each n and divided by unit area. All data just described are presented as the means ± SEM of n = 4. (F) Morphological characterization of endothelial cells that were cocultured with primary astrocytes showing nuclei stained with Hoechst (blue), IL-1β visualized with Alexa Fluor 488 (green) and the tight junction protein ZO-1 visualized with Alexa Fluor 594 (red). Magnification: 60x. Scale bar: 20 μm. The zoomed insets show the condition of the nuclei and the distributions of ZO-1 and IL-1β. DMSO = vehicle, Inh = β-secretase inhibitor IV, - or Glu refers to glutamate treatment. All experiments were performed in duplicate. (A–D) ANOVA followed by Tukey’s test and (E) Kruskal-Wallis test followed by Dunn’s test with Bonferroni correction. * P < 0.05, ** P < 0.01.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration

doi: 10.3389/fncel.2021.656832

Figure Lengend Snippet: BACE1-inhibited astrocytes reversed the increased IR and cytoplasmic distribution of the ZO-1 protein caused by glutamate in ECs. (A) FI of ZO-1 per unit area, quantified from 20 cells per treatment for each n ( n = 4 performed in duplicate) and divided by unit area. The data are presented as the means ± SEM. ANOVA followed by Tukey’s test. * P < 0.05. (B) Fluorescence profiles (right column) showing the cellular distributions of ZO-1 (red line) in relation to the nucleus (blue line) and IL-1β (green line) based on the representative images (left column). The black arrows indicate the tight junction peaks to show the changes in ZO-1 distribution by the treatment. The white line drawn on each cell represents a distance of 50 μm. DMSO = vehicle, Inh = β-secretase inhibitor IV, - or Glu refers to glutamate treatment.

Article Snippet: The first antibody mixture contained rabbit anti-GFAP (PA516291, Invitrogen, 1:250), mouse-anti-phospho-PHF-tau (MN1020, Thermo Fisher Scientific, 1:250) and DyLight 649-labeled Ulex Europaeus agglutinin (UAE) lectin (Vector Labs; DL-1068; 1:750), and the second antibody mixture contained anti-BACE1 (ab108394, Abcam, 1:100), mouse anti-GFAP (G3893, Sigma-Aldrich, 1:250) and DyLight 649-labeled UAE lectin.

Techniques: Fluorescence

Journal: Respiratory Research

Article Title: Mucus threads from surface goblet cells clear particles from the airways

doi: 10.1186/s12931-021-01898-3

Figure Lengend Snippet: Mucus bundles made by submucosal glands were transported by cilia. A Schematic drawing of the tilted table with heating to 37 °C where pig distal trachea and primary bronchi were mounted to ensure air–liquid interface and transport against gravity. B Image sequence from two sequential low-resolution time-lapses lasting five minutes each. Alcian blue stained mucus bundles (arrows) on explanted trachea from a weaned WT piglet. Speed of corresponding movies (Additional files and ) increased 16×. C Bundle thickness measured on the airway surface, each data point is the mean of at least ten measurements per bundle, median with interquartile range. WT: 3 piglets, 3 time-lapses, 8 bundles. CF: 3 piglets, 4 time-lapses, 11 bundles. D Explanted WT piglet airway mucus bundles stained with Alcian blue (blue), LTL (green) and merged low-resolution image. E Confocal high-resolution image of formalin-fixed paraffin section from a newborn piglet trachea stained with antibodies against MUC5B and MUC5AC, illustrating the production of MUC5B (green) in submucosal mucous cells and mostly MUC5AC (red) in surface goblet cells. Some MUC5B was observed in surface cells. F Surface goblet cell mucus stains with the UEA1 lectin (red), whereas mucus in submucosal glands (MUC5B) stains with the lectin LTL (green). G Explanted WT piglet airway with LTL stained mucus bundles, low-resolution image. Arrowheads indicate gland openings. H Bundle thickness measured on the airway surface compared to at the gland openings. The mean of ten measurements per opening is presented as one data point. Data presented as median with interquartile range. WT: 3 piglets, 9 time-lapses, 36 bundles. CF: 2 piglets, 3 time-lapses, 8 bundles. WT surface vs. WT opening P = 0.0025 **, Kruskal–Wallis and Dunn´s multiple comparisons test

Article Snippet: To visualize the different components of the mucus, tissue was stained with 0.4 mM Alcian blue 8GX pH 7.4 (Cat# A5268, Sigma-Aldrich, St. Louis, MO), 5 µg/ml fluorescein labeled Lotus tetragonolobus (LTL) lectin (Cat# FL-1321-2, Vector laboratories, Burlingame, CA) and/or 5 µg/ml DyLight 649 labeled Ulex europaeus agglutinin (UEA1) (Cat# DL-1068-1, Vector laboratories, Burlingame, CA) dissolved in oxygenated Krebs-glucose buffer, pH 7.4.

Techniques: Sequencing, Staining, Paraffin Section