fitc conjugated uea1 antibody  (Vector Laboratories)

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a – g , Representative immunofluorescence images of palatine tonsil sections from adult patients with OSA stained with the indicated antibodies and analyzed by confocal microscopy. a , Transition zone from the surface to the crypt epithelium. Stratified squamous epithelial cells at the surface (arrows) and UEA1 + epithelial cells infiltrated with CD3 + lymphocytes (arrowheads) at the transition area to the crypt epithelium are highlighted. DAPI, 4,6-diamidino-2-phenylindole. b , High-resolution analysis of the non-keratinized stratified squamous surface epithelium. PDPN-expressing basal cuboidal epithelial cells are highlighted (arrowheads). c , Morphology of the lymphoreticular crypt epithelium. Broadened PDPN-expressing basal layer of the crypt epithelium (arrows) and papillary extension providing access for blood vessels (asterisk) are highlighted. d , Epithelial and subepithelial (arrowheads) areas underpinned by PDPN + stromal cells. Boxed area shows the magnified epithelial–lymphoid tissue interface. e , B cell follicles with GCs and mantle zones (MZ) underpinned by PDPN + FRCs. f , CD3 + lymphocytes in epithelial, GC (asterisk) and interfollicular T cell (arrowhead) areas. g , T cell area surrounding B cell follicles (Fo) underpinned by PDPN + FRCs (arrowhead) and ACTA2 + PDPN − VSMCs (arrow). Square boxes show cell networks at higher magnification, and rectangular boxes indicate the regions of PDPN- and ACTA2-intensity measurements shown in h . h , Average pixel intensity of ACTA2 and PDPN signal-intensity measurements. The x axis represents the horizontal distance through the selection (rectangular boxes in g ), and the y axis represents the vertically averaged pixel intensity. The arrow highlights ACTA2 + PDPN + myofibroblasts in the perivascular space. Microscopy images are representative for n = 3 adult patients with OSA.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Selection, Microscopy

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a - f , Representative immunofluorescence images of palatine tonsil sections from adult patients with OSA stained with the indicated antibodies and analyzed by confocal microscopy. a , Structure of the palatine tonsil. UEA1 staining of the lymphoreticular epithelium highlights deep crypts permeating palatine tonsils. b , d , High resolution analysis of the non-keratinized stratified squamous surface epithelium. PDPN-expressing basal cuboidal epithelial cells are highlighted (arrowheads). c , e , Morphology of the lymphoreticular crypt epithelium. Broadened PDPN-expressing basal layer of the crypt epithelium is highlighted (arrows). f , Lymphoreticular crypt epithelium occupied by CD11c + myeloid cells. Boxed area shows magnified intraepithelial CD11c + cells surrounding a blood vessel (asterisk). Microscopy images are representative for n = 3 adult patients with OSA.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Microscopy

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , Representative UMAPs of distinct tonsillar stromal cell types according to the PhenoGraph clustering algorithm based on forward scatter (FSC)-A, side scatter (SSC)-A, CD31, ACTA2, PDPN and UEA1 flow cytometry data of CD45 − CD235a − cells ( n = 3 pediatric and n = 3 adult patients with OSA). Epithelial cells (EpCs), ACTA2 + cells, BECs, LECs, FRCs and negative cells (N) are indicated. b , UMAPs show the expression pattern of the indicated markers. c – e , Quantification of the indicated stromal cell types as a percentage of CD45 − CD235a − live cells in pediatric ( n = 5) and adult ( n = 6) patients with OSA. Data show average values of left and right tonsils for each patient. Mean and s.d. are indicated. P values were calculated with the two-sided Mann–Whitney test.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Nature Immunology

Article Title: PI16 + reticular cells in human palatine tonsils govern T cell activity in distinct subepithelial niches

doi: 10.1038/s41590-023-01502-4

Figure Lengend Snippet: a , b , PI16-positive cells in the subepithelial niche of tonsils from adult patients with OSA or tonsillitis. Cryosections were stained with the indicated antibodies and analyzed by confocal microscopy. c , High-resolution analysis of PI16 + cells shows intracellular PI16 signal in FBLN1 + reticular cells. Sections were stained with anti-FBLN1 and anti-PI16, and data were acquired by confocal microscopy and reconstructed in 3D. d , Morphology of the FBLN1 + subepithelial compartment in adult tonsils. Histological sections were stained with the indicated antibodies, and data were acquired and analyzed by confocal microscopy. The boxed area shows an FBLN1 + reticular network around UEA1 + endothelial cells at higher magnification on the right. e , High-resolution reconstruction of the adult subepithelial FBLN1 + FRC niche occupied by CD3 + and CD20 + lymphocytes. Sections were stained with the indicated antibodies, and data were acquired by confocal microscopy and reconstructed in 3D. f , Surface contact areas (blue) of FBLN1 + cells with CD20 + (arrows) and CD3 + (arrowheads) cells are shown at higher magnification. Microscopy images are representative for n = 3 adult patients with OSA and n = 3 adult patients with tonsillitis.

Article Snippet: Single-cell suspensions of tonsillar stromal cells were prepared as described above, incubated with Fixable Viability Stain 510 (1:1,000, BD Biosciences) and subsequently stained for 20 min at 4 °C in PBS containing 2% FCS and 2 mM EDTA with the FITC-conjugated lectin UEA1 (1:100, Sigma) and the following fluorochrome-conjugated antibodies: anti-human PDPN, anti-human CD45, anti-human CD235a and anti-human CD31.

Techniques: Staining, Confocal Microscopy, Microscopy

Journal: eLife

Article Title: Multi-targeted therapy resistance via drug-induced secretome fucosylation

doi: 10.7554/eLife.75191

Figure Lengend Snippet: Representative snapshots from wide-field fluorescence images of indicated tumor tissues from mice as in stained for fluorescein-conjugated AAL (core fucosylation; green), Alexa Fluor 594-conjugated UEA1 (terminal fucosylation; red), RCAS1 (golgi marker; orange), and DAPI (nuclei; blue). Scale bar indicates 75 μm. Beside shows fluorescence quantification of at least 50 regions-of-interest (ROIs) within the tumor mass evaluated per FFPE tumor slice. Values indicate relative fluorescence intensity (RFI) relative to PBS (means ± SD of at least five FFPE tumor slices from four to six biological replicates). For statistical analysis, a two-tailed, unpaired Student’s t-test with Welch’s correction for unequal variance was used.

Article Snippet: For lectin fluorescent staining of cells and paraffin sections, we used 15 μg/mL fluorescein-labeled AAL (Vector Laboratories) or 4 μg/mL FITC- or Alexa Fluor 594-conjugated UEA1 (Thermo Scientific) according to manufacturer’s protocol and following standard immunofluorescence protocols.

Techniques: Fluorescence, Staining, Marker, Two Tailed Test