biotinylated uea1 lectin  (Vector Laboratories)

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , Uniform Manifold Approximation and Projection (UMAP) of 22,932 CD45 − thymic cells from 2-mo and 18-mo female C57BL/6 mice, annotated by cell type subset and outlined by cell compartment (epithelial; fibroblast; endothelial; MEC; vSMC/PC; nmSC). b , ThymoSight integration of public data for murine nonhematopoietic thymic stromal cells, including our own dataset ( n = 297,988) annotated by publication source and outlined by cell type and compartment. c , Violin plots highlighting key genes marking individual subsets within individual structural compartments (fibroblast, endothelium and epithelium). d , e , UMAPs of individual structural compartments color-coded by cell type subset ( d ) and age cohort ( e ). n EC = 1,661; n FB = 13,240; n TEC = 6,175. f , Scaled change in frequency for each individual structural cell subset with age. g , Gating strategy and quantities for cell populations within the epithelial lineage (based on previous work ) in 2-mo ( n = 10) and 18-mo ( n = 10) mice. First, based on a CD45 − EpCAM + parent gate, tuft cells were identified by expression of L1CAM, then all other TECs were assessed for expression of conventional TEC markers UEA1 and Ly51. Within the UEA1 hi Ly51 lo mTEC population CD104 + MHCII lo cells were identified as mTEC1. Cells that were deemed as non-mTEC1 were then fractionated based on MHCII and Ly6D. h , Concatenated flow cytometry plots and graphs highlighting the frequency of Ly51 − UEA1 − (DN-TECs) across lifespan (gated on CD45 − EpCAM + MHCII + cells). i , Violin plots of aaTEC1 and aaTEC2 novel markers. j , k , Flow cytometry plots ( j ) and quantities ( k ) for aaTEC1 and aaTEC2 populations in 2-mo ( n = 15), 12-mo ( n = 10) or 18-mo ( n = 13) male and female C57BL/6 mice. Summary data represent mean ± s.e.m.; each dot represents an individual biological replicate. Statistics were generated using a two-tailed Mann–Whitney test comparing within individual subsets ( g ) or Kruskal–Wallis ( k ) test with Dunn’s correction.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Expressing, Flow Cytometry, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a–c , Concatenated flow cytometry plots and quantities for cell populations within the fibroblast ( a ), endothelial ( b ), and “other” ( c ) cell lineages in 2-mo (n = 10) and 18-mo (n = 10) mice. c , Concatenated flow cytometry plots and quantities for pericytes (PC), vascular smooth muscle cells (vSMC) and mesothelial cells (MEC) (n = 10/age). d , Frequency and numbers of DN-TEC across lifespan: 2-mo (n = 14), 6-mo (n = 5), 9mo (n = 15), 12-mo (n = 5), and 18+mo (n = 18). e , Violin plots with extensive list of aaTEC1 and aaTEC2 markers. f , Gating strategy for aaTECs. aaTEC1 were first gated on CD45 − TER119 − then PDGFRα - CD31 − cells. EpCAM + MHCII + cells were gated as the whole TEC compartment, then mTECs and cTECs were excluded by taking the UEA1 − Ly51 − double negative fraction and gating on CLDN3. aaTEC2 were also first gated on CD45 − TER119 − then PDGFRα - CD31 − cells. EpCAM − MHCII + cells were then gated and PDPN + PDGFRβ - were classed at aaTEC2. Summary data represents mean ± SEM; each dot represents an individual biological replicate. Statistics for a–c were generated using two-tailed Mann–Whitney tests comparing within individual populations and for d using the Kruskal–Wallis test with Dunns correction.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Flow Cytometry, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , Representative flow cytometry plots from 12–18-mo male and female Foxn1 nTnG mice at the indicated ages, gated on tdTom − GFP + cells. tdTom − GFP + EpCAM + cells were then assessed for expression of the conventional TEC markers UEA1 and Ly51. b , Claudin-3 and UEA1 expression on tdTom − GFP + EpCAM + cells in Foxn1 nTnG mice, and quantification of claudin-3 on UEA1 + mTEC and UEA1 − Ly51 − DN-TECs ( n = 4 biological replicates representing individual mice). c , Podoplanin (Pdpn) and PDGFRa expression on tdTom − GFP + EpCAM − cells ( n = 4 biological replicates representing individual mice). d , Number of GFP + EpCAM + UEA1 − Ly51 − Cldn3 + aaTEC1 and GFP + EpCAM − PDPN + PDGFRα − aaTEC2 cells in 2-mo ( n = 6) and 18-mo ( n = 4) mice. e , scRNA-seq was performed on CD45 − cells isolated from male and female 20-mo Foxn1 tdTom and age-matched WT mice, and integrated into the epithelial data described in Fig. . UMAP of 8,505 cells of the epithelial compartment in the integrated data showing the TEC annotated subsets (top) and overlaid expression of tdTomato (bottom). Scale represents log-transformed average expression of the tdTomato-WPRE element. f , RNA velocity on selected TEC populations in 2-mo (top) or 18-mo (bottom) mice. n 2mo = 1,989; n 18mo = 3,382. g , Vein plots describing the continuous transition of 18-mo early prog , mTEC1, mTEC prol and aaTEC subsets to their predicted descendants (represented by diagonal flows) and the dynamic relative frequencies (vein width on the y axis) of these TEC subsets in the thymus over the binned pseudotime. h , Expression of thymocyte markers Thy1 and Lck overlaid on the 18-mo spatial transcriptomics dataset. Outline represents thymocyte-poor area overlaid onto heatmap showing aaTEC1 or aaTEC2 signatures. i , Two representative images in 12–18-mo male and female Foxn1 nTnG mice showing tdTomato and GFP expression with HD-TEC areas highlighted, with few or no tdTomato + cells. Scale bar, 50 μm. j , Human tissue sections from a 50-year-old woman. Shown are consecutive sections with H&E, cytokeratin or CD1a staining. k , aaTEC1 and aaTEC2 gene signatures (top 20 marker genes from our mouse data converted to human orthologs; Supplementary Fig. and Supplementary Table ) were overlaid on human thymic epithelial cells ( n TEC = 40,144) from single-cell sequencing datasets generated and published elsewhere , , . Summary data represents mean ± s.e.m. and each dot represents an individual biological replicate. Statistics were generated ( b – d ) using a two-tailed Mann–Whitney test.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Flow Cytometry, Expressing, Isolation, Transformation Assay, Staining, Marker, Sequencing, Generated, Two Tailed Test, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Age-related epithelial defects limit thymic function and regeneration

doi: 10.1038/s41590-024-01915-9

Figure Lengend Snippet: a , 3D reconstruction and representative images of high-density TEC region from 12-mo Foxn1 nTnG mice stained with DCLK1 or UEA1 to highlight tuft cells and M-like cells, respectively. b , Flow cytometry plots showing proportion of selected mimetic cells (tuft, corneocyte and M-cells) in 2-mo (n = 6) and 18-mo (n = 8) Foxn1 nTnG mice. Mimetic cells were first gated on EpCAM + GFP + cells, then mTECs (UEA1 hi Ly51 lo ) were assessed for the mimetic cell markers DCLK1 (tuft cells), GP2 (microfold cells), and Ly6D (corneocytes). Bar graph shows quantification of mimetic cell numbers. c , Flow cytometry plots showing mimetic cell frequency in 18-mo Foxn1 nTnG mice (n = 8) gated on EpCAM + GFP + UEA1 − Ly51 − DN-TECs. Bar graph shows quantification of mimetic cells comparing mTECs (as in Extended Data Fig. 7b) and DN-TECs. d , Aire and Foxn1 expression in TEC subsets. e , Representative confocal images of thymic sections from 12-mo Foxn1 nTnG mice stained with anti-AIRE, with the medulla or high-density TECs highlighted. Scale bar: 50μm. Summary data represents mean ± SEM; each dot represents an individual biological replicate. Statistics for b-c were generated using two-tailed Mann–Whitney tests comparing within individual mimetic cell subsets.

Article Snippet: The antibodies used were rabbit anti-pan-cytokeratin (Dako, cat. no. Z0622), anti-K5 (BioLegend, cat. no. poly19055), rat anti-mouse K8/18 (Troma-1; Developmental Studies Hybridoma Bank), rabbit anti-K14 (Abcam, cat. no. EPR17350), rat anti-mouse AIRE (WEHI, clone 5H12), rabbit anti-human/mouse DCLK1 (LSBio, cat. no. LS-C100746) and biotinylated UEA1 lectin (Vector Labs, cat. no. B-1065).

Techniques: Staining, Flow Cytometry, Expressing, Generated, Two Tailed Test, MANN-WHITNEY

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: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, Paraffin Section

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 threads were secreted by surface goblet cells. A Image sequence from a low-resolution time-lapse where beads were collected by threads on an explanted WT piglet trachea. Arrow points to moving mucus assemblies and arrowhead to long mucus formation with increasing thickness. Speed of corresponding movie (Additional file ) increased 16×. B The lectin UEA1 (blue) stained bead-collecting assemblies (red), low-resolution image of explanted WT piglet trachea. C Low-resolution image of explanted live UEA-stained (red) WT piglet trachea. Yellow arrows indicate surface goblet cells and white arrows point to threads secreted from the goblet cells. D High-resolution Airyscan Z-stack of explanted live WT piglet trachea stained with Syto 59 (DNA, grey) and UEA1 (mucus, red). Yellow arrows indicate surface goblet cells and white arrows point to threads secreted from the goblet cells. E Scanning electron micrographs from three different WT piglet tracheas. Threads from secreting goblet cells (sGC) are indicated by green arrows. Some goblet cells (GC) were not secreting. F Scanning electron micrograph of mucus bundle originating in a submucosal gland. Note that the mucus bundle is thicker than the mucus threads in E . Sample from a CF piglet trachea. G Comparison of mucus bundle and mucus thread thickness in scanning electron micrographs. One data point represents the mean of 10 measurements per bundle or thread. Mucus threads were thinner than bundles in both WT and CF, Mann–Whitney test: WT bundles vs. WT threads P < 0.0001 ****, CF bundles vs. CF threads P = 0.0022 **. WT bundles: median 5.9 µm, 6 bundles (4 pigs), WT threads: median 0.47 µm, 13 threads (4 pigs), CF bundles: median 3.1 µm, 6 values (4 pigs), CF threads: median 0.46 µm, 6 values (4 pigs). Data presented as median with interquartile range. H The thread median thickness was 0.5 µm after fixation

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY

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 threads from surface goblet cells were thinner than mucus bundles. A Image sequence from a low-resolution time-lapse where threads collected beads on an explanted WT piglet trachea. Speed of corresponding movie (Additional file ) increased 16×. Mucus bundle coming out of a gland opening indicated by light blue arrowhead. Dark blue arrow: immobile bundle. Yellow, green and purple arrows indicate different moving mucus assemblies. B Low resolution image of bundles (LTL, blue arrow) and beads gathered into threads (yellow arrow) on WT piglet tracheas (arrowhead: gland opening). C Low-resolution image of explanted WT piglet trachea with bundle from a submucosal gland, LTL (green) and threads, UEA1 (red). D Airyscan high-resolution Z-stack of LTL-stained bundles (green) from submucosal glands and UEA1-stained threads (red) in a live explanted WT piglet trachea. E Thickness analysis of mucus bundles (LTL, green) and mucus threads (UEA1, red) in the image in D . Inset: zoom of the threads in the image center. F Mucus bundle thickness was calculated as a mean of 10 measurements close to the gland opening (1A, 2A) and further along the bundle (1B, 2B), as indicated in ( E ). Bundles were thicker close to the gland opening, Mann–Whitney test: 1A vs. 1B P < 0.0001 ****, 2A vs. 2B P = 0.0052 **. Thread thickness was calculated in the same way, as indicated in ( E ). Based on bundle thickness in live explants compared to fixed scanning electron micrographs, the maximum mucus thread thickness was determined to 2 µm (dashed line). Thread T3 was thicker than T4, Mann–Whitney test P < 0.0001 ****. Bundles were thicker than threads, Mann–Whitney test P = 0.0095 **. Data presented as median with interquartile range. G Scanning electron micrograph from a WT piglet trachea with mucus bundle from a submucosal gland (blue arrowhead). H Scanning electron micrograph of WT piglet trachea mucus thread (yellow arrows), GC: goblet cell, sGC: secreting goblet cell

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY

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 threads collected together and on mucus bundles. A Image sequence from a high-resolution video illustrating a UEA1-positive mucus assembly (green arrow) docking to an LTL-positive bundle (blue arrow) coated with UEA1 on a newborn WT piglet trachea (Additional file ). B Image sequence from a high-resolution video illustrating a UEA1-positive mucus assembly (green arrow) docking to another UEA1-positive mucus assembly (dark green arrow) on a newborn WT piglet trachea (Additional file ). C Bead-gathering mucus formations were divided into threads and mucus assemblies based on the maximum thread thickness of 2 µm determined by the difference in thickness between hydrated and fixed mucus. One thread was observed in WT and none in CF. WT: 15 values (9 pigs), CF: 6 values (3 pigs). D Thickness measurements of UEA1-stained mucus in high-resolution images from live explant tracheas from weaned pigs, newborn WT and CF piglets resulted in 3 threads and 48 mucus assemblies (6 weaned pigs), 3 threads and 54 mucus assemblies (10 newborn WT piglets), 2 threads and 37 mucus assemblies (12 newborn CF piglets), using the same criteria as in C. Mucus assemblies in weaned pigs were thicker than the corresponding assemblies in newborn CF piglets, P = 0.0418 * Kruskal–Wallis and Dunn´s multiple comparisons test. E Alcian blue stained mucus bundle velocity in weaned, newborn WT and CF tracheas measured in low-resolution time-lapses recorded on the tilted table. Each data point represents one pig. Weaned 19 pigs, newborn WT 14 pigs and newborn CF 12 pigs. Weaned bundles were faster than newborn WT bundles, P = 0.0111 *, Mann–Whitney test. F There was no difference in velocity between bead-collecting mucus assemblies in newborn WT and CF piglet trachea. WT: 15 values, 15 time-lapses, 8 pigs. CF: 12 values, 12 time-lapses, 6 pigs. Mann–Whitney test: P > 0.05. Data in ( C – F ) presented as median with interquartile range

Article Snippet: Biotinylated Lotus tetragonolobus (LTL) lectin (Cat# B-1325-2, Vector Laboratories, Burlingame, CA) or biotinylated Ulex europaeus Agglutinin I (UEA1) lectin (Cat# B-1065-2, Vector Laboratories, Burlingame, CA) was incubated in blocking solution for one hour at ambient temperature on dewaxed and rehydrated slides.

Techniques: Sequencing, Staining, MANN-WHITNEY

Journal: Histochemistry and Cell Biology

Article Title: Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage

doi: 10.1007/s00418-016-1453-4

Figure Lengend Snippet: Guca2a and Guca2b expression by cells of the secretory lineage in the duodenum and colon. a Guca2a expression in Paneth cells in duodenum. b Guca2a expression in cells at the base and the neck region of the crypts in colon. c Guca2a expression in a duodenal brush cell. d Guca2a expression in a duodenal goblet cell. e Costaining of Guca2a transcript ( dark brown ) and lectin UEA1-binding fucose glycoproteins ( bright red ) in duodenum. f Guca2b expression in Paneth cells in duodenum. g Guca2b expression in cells at the base of the crypts in colon. h Guca2b expression in a duodenal brush cell. No Guca2b transcript was observed in goblet cells. i , j Guca2b levels were generally low in columnar cells, but comparatively high in columnar cells adjoining goblet cells. B brush cell, G goblet cell, P Paneth cell

Article Snippet: For detection of fucose glycoprotein-producing cells, sections used for RNAscope were re-hydrated in PBS, treated with a biotin blocking reagent (Dako), and incubated (1 h, at room temperature) with biotinylated lectin UEA1 (Vector Labs).

Techniques: Expressing, Binding Assay