rat anti mouse lyve1  (R&D Systems)

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 1 | Junctional heterogeneity in capillary LECs. a, Whole-mount immunofluorescence of ear skin from a 25-week-old Cdh5-GFP mouse expressing VE-cadherin-GFP fusion protein (VE-cad). Boxed areas magnified below show unsegmented (arrows) and focal (arrowheads) VE-cadherin+ junctions in lymphatic capillary (left) and precollecting vessel (middle), and continuous zipper junctions in LYVE1− collecting vessel (right). b, Immunofluorescence in 12-week-old mouse ear skin showing VE-cadherin colocalization with CLDN5 at junctions. Line intensity profiles through lines 1 and 2 of respective stainings are depicted. c, Immunofluorescence at the indicated ages depicting junctional heterogeneity. Boxed areas are magnified. d,e, Quantification of lymphatic vessel sprouting (percentage of spiky ends of all lymphatic capillary ends, n = 7, 12 per respective stage, mean ± s.e.m.; d) and LEC proliferation (percentage Ki67+ of all LECs by flow cytometry, n = 3, 6, 8, 6 mice per respective stage, mean ± s.e.m.; e). f, Whole-mount silver nitrate (Ag) staining of ear dermis showing deposits around cell perimeter, including the lobe tips (arrow),

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Immunofluorescence, Expressing, Flow Cytometry, Staining

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 2 | Morphology and remodelling of intercellular overlaps between capillary LECs. a, Constructs for mosaic multicolour labelling of LECs using membrane-localized fluorescent proteins. b, Whole-mount immunofluorescence of mosaically labelled dermal LECs in a 6-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse after 4-OHT treatment at 3 weeks, showing lobate shape in LYVE1+ capillaries and elongated shape in LYVE1− collectors (arrowheads). c, Whole-mount immunofluorescence of embryonic back skin (E17) or ear skin at indicated postnatal stages in iMb2-Mosaic;Vegfr3-creERT2 mice. d–f, Dermal LEC and vessel parameters, represented as mean ± s.d.: cell size (d, n = 24, 17 and 20 cells per respective stage), lobe number (e, n = 24, 48 and 20 cells per respective stage) and average lymphatic capillary width (f, n = 7, 5 and 9 mice per respective stage). Ordinary one-way ANOVA. g, Immunofluorescence of ear skin of a 12-week-old iMb2-Mosaic;Vegfr3-creERT2 mouse showing LYVE1 at LEC overlaps, with corresponding intensity plot. h, Double staining for cell surface and total LYVE1 (left), or with intradermally

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Construct, Membrane, Immunofluorescence, Double Staining

Journal: Nature

Article Title: Dynamic cytoskeletal regulation of cell shape supports resilience of lymphatic endothelium.

doi: 10.1038/s41586-025-08724-6

Figure Lengend Snippet: Fig. 3 | Cytoskeletal organization in lobate capillary LECs. a, Dot plot showing differential expression of cytoskeletal genes between capillary and collecting vessel LECs. Dot size illustrates percentage of cells with transcript counts, colour illustrates average expression (log2-fold difference). Cap, lymphatic capillary; Col, collecting vessel. b, Whole-mount immunofluorescence of adult ear skin showing microtubule network in dermal capillary LECs. Cell outline, based on VE-cadherin and LYVE1 staining, in red. c, Quantification of microtubule (MT) anchoring and density in capillary LECs in 9–12-week-old mice. Cell outline from c in green, with MT endpoints shown by yellow (concave) and purple (convex) dots. Data represent the percentage of MT anchoring (left; n = 5 LECs from five mice, 20–53 MT per cell), or MTs per µm of cortex in concave (right; n = 156 MTs, 5 LECs from five mice) versus convex (n = 56 MTs, 5 LECs

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used The following antibodies were used for whole mount immunofluorescence (dilution 1:100-1:500): chicken anti-GFP (ab13970, Abcam), goat anti-mouse VEGFR3 (AF743, R&D Systems), goat anti-mouse VE-cadherin (R&D Systems, AF1002), goat anti-mouse PECAM1 (R&D, Systems AF3628), mouse anti-HA tag, Alexa Fluor 647 (Cell Signalling Technology, 6E2), rabbit anti-alpha tubulin (Abcam, ab52866), rabbit anti-GFP (A11122, Thermo Fisher Scientific), rabbit anti-DsRed (Takara Bio, 632496), rabbit anti-mouse LYVE1 (Reliatech, 103-PA50AG), rabbit anti-mouse CLDN5 (Invitrogen, 34-1600), rat anti-mouse PECAM1 (553370, BD Pharmingen), rat anti-mouse LYVE1-Alexa FluorTM 488, Clone ALY7 (Invitrogen, 53-0443-82), rat anti-mouse LYVE1 (R&D Systems (MAB2125), rat Anti-Mouse CD29 Clone 9EG7 (BD Pharmingen, 553715) All secondary antibodies were conjugated to Cy3(JIR, 712-165-153),(JIR, 711-166-152), Dylight 405 (JIR, 712-475-153), Alexa Fluor 488 (JIR, 703-545-155), (JIR, 712-545-153), (JIR, 711-545-152), Alexa Fluor 594 (JIR, 705-585-147), Alexa Fluor 647(JIR, 712-605-153),(JIR, 705-605-147),(JIR, 711-605-152) or Alexa Fluor 680 (JIR, 705-625-147) were raised in donkey and obtained from Jackson ImmunoResearch(JIR) .

Techniques: Quantitative Proteomics, Expressing, Immunofluorescence, Staining

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: ( A ) VE-cadherin (red) and LYVE1 (green) staining of mouse ear pinna dermis. A flattened overview image and zoom-in of a single optical slice are shown. The images represent n = 3 mice. ( B ) The dot plot shows mean ± SD percentage of wild-type or CCR7 −/− DCs that detached, subsequent to the arrest at multicellular junction. The data point represents n = 6 wild-type and n = 5 CCR7 −/− biological replicates from three independent experiments, altogether, representing 401 wild-type and 140 CCR7 −/− DCs. The data is related to Fig. . ( C ) Schematic shows mCherry tagged full-length CCL21 and CCL21ΔC-mCherry that lacks the charged C-terminus (Hirose et al, ). ( D ) Quantification of wild-type DC transmigration sites in CCL21ΔC-mCherry expressing LEC cultures. The stacked bar graph shows transmigration sites as a percentage of all events from 15 biological replicates in three independent experiments and, altogether, n = 128 transmigration events. ( E – I ) The bar graphs show the mean percentage ± SD of DCs positive for ( E ) CD86, ( F ) CD11b, ( G ) CD11c, and ( H ) MHCII in wild-type versus CCR7 −/− DCs. In ( I ) the bar graph shows mean cell size ± SD of wild-type vs CCR7 −/− DCs normalized to the average of wild-type DCs, which was set at 1, in each experiment. In ( E , I ) The data points represent n = 4 biological replicates/genotype in two independent experiments. The number of analyzed DC singlets was at least 277000/sample. ( J ) A capture of a phase contrast/immunofluorescence microscopy showing wild-type (magenta) and CCR7 −/− DC (green), on a LEC monolayer. The images represent n = 3 biological replicates. ( K , L ) Western blot panel of non-muscle myosin heavy chain 2 A (NMH-IIA), actin, and HSC70 (for loading control) in wild-type versus CCR7 −/− DCs. Quantification of the band intensities is shown in the western blot data shown in ( L ). The bar graph in ( L ) shows, mean intensity ± SD. Data was normalized to the average of wild-type DCs, which was set at 1. The data in ( K , L ) represent n = 4 biological replicates/genotype and two independent experiments. ( M ) Quantification of human CCL21 mRNA levels in human specific siCCL21 transfected and mouse CCL21ΔC-mCherry expressing LECs. The dot plot shows the mean hCCL21 mRNA level ± SD normalized to the average of siControl samples, which was set at 1 (green line), in each experiment. Data points represent n = 4 biological replicates/ siRNA oligo in 2 independent experiments. P -values show the comparison to controls. ( N ) Images show mouse CCL21ΔC-mCherry expression in siControl or human CCL21-specific siCCL21 oligo transfection. Images represent n = 4 biological replicates in 2 independent experiments. ( O ) Quantification of transmigration sites of wild-type DCs in siControl or siCCL21 transfected and CCL21-mCherry or CCL21ΔC-mCherry expressing LEC cultures. Transmigration sites are shown as a percentage of all events from 2 independent experiments, consisting of siCTRL01 n = 209 transmigration events (3 biological replicates), siCTRL02 n = 187 (3 biological replicates), siCCL21-05 n = 137 (3 biological replicates), or siCCL21-08 n = 249 (4 biological replicates) transfected and CCL21-mCherry expressing LEC cultures and of siCTRL01 n = 96 (4 biological replicates), siCTRL02 n = 68 (4 biological replicates), siCCL21-05 n = 69 (4 biological replicates), or siCCL21-08 n = 223 (8 biological replicates) transfected and CCL21ΔC-mCherry expressing LEC cultures. ( P ) The bar graph shows a mean number of observed CCL21ΔC-mCherry exocytosis events/cell ± SD in control or BAPTA-AM treated LECs. The data points represent n = 37 DMSO control and n = 33 BAPTA-AM treated cells in 11 biological replicates across two independent experiments. Data information: In ( A ), yellow arrows indicate the site of zoom-in image and white arrowheads indicate the multicellular junctions. In ( J ), the white arrowheads indicate wild-type and yellow arrows CCR7 −/− DC dendrites. The p -values in ( B ), ( E – I ), and ( M ) were calculated using a parametric T-test with Welch’s correction, whereas in ( L ) and ( P ) p -values were calculated using the Mann–Whitney test. In ( O ), Chi-square test was used to test the significance of data. The scale bar is 20 µm in overview images ( A and J ), 5 µm in zoom-in images ( A ), and 50 µm in ( N ).

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Staining, Transmigration Assay, Expressing, Immunofluorescence, Microscopy, Western Blot, Control, Transfection, Comparison, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions

doi: 10.1038/s44318-024-00129-x

Figure Lengend Snippet: Figure 2 is shown with alternative colors (non-red and -green) in Appendix Fig. . ( A ) LEC monolayer expressing CCL21ΔC-mCherry (red) and stained for VE-cadherin (magenta) and nuclei (DAPI, blue). The data in ( A ) represents at least n = 3 independent experiments. ( B , C ) A capture of immunofluorescence live recording (Movie ) shows primary LEC monolayer expressing CCL21ΔC-mCherry (red) and stained for VE-cadherin (green). Exocytosis events were analyzed within a 7 µm wide region. ( C ) A histogram showing exocytosis events (mean number of secretions/cell + SD) at the LEC junctions, as a function of distance (in µm) from the nearest multicellular junction. The data in ( B and C ) represents 241 secretion events from n = 22 LECs in 5 biological replicates across two independent experiments. ( D – F ) LECs expressing chemokine CCL21ΔC-mCherry (red) and the indicated EGFP-tagged RAB-GTPase (green). The nuclei are stained with DAPI (blue). The images are representative of n = 3 biological replicates in three independent experiments. Quantification in ( E ) shows percentage of CCL21ΔC-mCherry+ vesicle colocalization with the indicated EGFP-RAB GTPases in the whole LEC area. The dot plot shows the mean percentage ± SD. Each data point represents a single analyzed cell (EGFP-RAB3D ( n = 20); EGFP-RAB27A ( n = 18); EGFP-RAB37 ( n = 25) and EGFP-RAB6A ( n = 24)), from a total of 3 biological replicates in three independent experiments. The histogram ( F ) shows the distribution (mean percentage) of the colocalized vesicles as a function of distance from a multicellular junction. The number of samples was the same as in ( E ). ( G ) A TNF-α-treated LEC, expressing chemokine CCL21ΔC-mCherry (red) and, stained for endogenous CCL2 (green), and nuclei (DAPI, blue). The images represent n = 2 independent experiments. ( H ) A LEC expressing chemokine CCL21ΔC-mCherry (red) and stained for endogenous RAB6 (green). The images represent n = 2 independent experiments. ( I ) A TNFα-treated LEC monolayer stained for endogenous CCL2 (red), RAB6 (green), and nuclei (DAPI, blue). The images represent n = 2 independent experiments. ( J ) Mouse-ear pinna dermis stained for CCL21 (red), RAB6 (green), LYVE1 (magenta), and nuclei (DAPI, blue). For clarity, the overview image shows only staining of LYVE1+ and nuclei. The images are representative of 3 mice. ( K ) Representative images show a mouse ear pinna dermis lymphatic pre-collector (continuous junctions, quantified in ( M , N ) and ( L ) capillary (discontinuous junctions, quantified in ( O , P ) stained for CCL21 (red), RAB6 (green), VE-cadherin (magenta). The overview images show VE-cadherin-only or CCL21 and RAB6. The zoom-in images show images of peripheral and perinuclear areas of the LEC. Figures 2K and L are shown with more examples in Appendix Fig. . ( M ) Quantification of CCL21 vesicle colocalization (mean percentage ± SD) with RAB6, in LECs showing continuous junctions in mouse ear pinna dermis in vivo. Each data point represents a single analyzed cell. ( N ) The histogram shows the distribution (mean percentage + SD) of CCL21 and RAB6 colocalized vesicles and non-colocalized CCL21 vesicles, as a function of distance from the multicellular junction (for LECs displaying continuous junctions). In ( K ) and ( M , N ), n = 29 cells representing 6 mice. ( O ) Quantification of CCL21 vesicle colocalization (mean percentage ± SD) with RAB6, in LECs showing discontinuous junctions in mouse ear pinna dermis in vivo. Each data point represents a single analyzed cell. ( P ) The histogram shows the distribution (mean percentage + SD) of CCL21 and RAB6 colocalized vesicles and non-colocalized CCL21 vesicles, as a function of distance from the VE-cadherin stained cell border (for LECs displaying discontinuous junctions). In ( L ) and ( O , P ), n = 15 cells representing 3 mice. Data information: In ( A ), ( D ), ( G – I ), and ( J – L ), the yellow arrow indicates the peripheral, and the white arrowheads the perinuclear area shown in the zoom-in image. The cell borders in ( D ), ( G – I ), and ( K , L ) are marked with white dotted lines. The cyan arrow shows an example of colocalization and the magenta arrowheads examples of non-colocalizing vesicles. In ( B ), the yellow arrow indicates LEC multicellular junction (shown in the zoom-in image and in Movie ). The vesicles that were exocytosed are marked with white arrowheads. The p -value in ( E ) was calculated using one one-way ANOVA test. Scale bars in ( A , B ), ( D ), and ( G – J ) are 20 µm in the overview images and 5 µm in the zoom-in images. In ( K , L ) the scale bars in overview images are 10 µm and 2 µm in the zoom-in images. .

Article Snippet: The used primary antibodies were: a mix of rat anti-mouse VE-cadherin (Biolegend, 138003; dilution of 1:100) and rat anti-mouse VE-cadherin (Invitrogen, 14-1441-81; 1:100) (Figs. and ; Appendix Fig. ); goat anti-mouse CCL21/6Ckine biotinylated antibody (R&D BAF457; 1:300) (Fig. and Appendix Fig. ); rabbit anti-RAB6 (D37C7, Cell signaling, 9625S; 1:100) (Fig. ; Appendix Fig. ); rat anti-mouse LYVE1 (R&D, MAB2125; 1:300) (Fig. ); rabbit anti-mouse LYVE1 (a kind gift from Dr. Kari Alitalo lab (Karkkainen et al, ); 1:1000) (Fig. ).

Techniques: Expressing, Staining, Immunofluorescence, In Vivo

Journal: Nature immunology

Article Title: An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut resident plasma cells

doi: 10.1038/s41590-022-01413-w

Figure Lengend Snippet: a, Immunofluorescence of IgA, GPR183, LYVE1 and DAPI on duodenum sections from a Gpr183GFP/+ reporter mouse. Scale bar is 100μm. b, Frequency of GPR183 in lamina propria of Gpr183GFP/+ reporter mouse measured by flow cytometry. c, Relative migration of lamina propria plasma cell and CD45+ in response to different concentrations of 7α,25-HC. d, Relative migration of lamina propria PCΔGpr183 and wild type at the indicated concentration of GPR183 ligand. Number of migrated cells were analyzed by flow cytometry and the relative migration was measured as fold of changes. e, Representative flow cytometry plot and percentage of IgA secreting PCs (CD98hiIgA+) in PCΔGpr183 and control mice, fed for one week with NF or HCF. f, Number of total IgA+ PC in duodenum of mice from (e). g, Representative IgA ELISPOT and compiled quantification of secreting IgA+ PCs from PCΔGpr183 and controls mice at steady state. h, Area and intensity quantification of IgA+ spots from (g). Each dot represents single IgA+spot. i, Experimental model illustrating the development of Tamoxifen inducible bone marrow (BM) retroviral chimera mice. BM cells from Rosa26-STOP-tdtomato Gpr183fl/fl and Rosa26-STOP-tdtomato Gpr183Wt were transduced with retroviral vector encoding for ERT2-cre. BM cells (10X106) were injected in irradiated C57/BL6 mice. j, 8 weeks after BM reconstitution mice were injected with Tamoxifen and 100μg of anti-MadCam1 for one week and euthanized for flow cytometry analysis of IgA+ PCs. CD98 PCs were previously gated on tdTomato+ cells. The results were pooled from three independent experiments (a and b)( n=6 mice); (c)(total of n=3 mice ); (d)(n=6mice for PCWt and n=3 mice for PCΔGpr183); (e)(n=5 mice for NF fed mice n=8 for HCF fed mice); (g)(n=5 mice per group); (h)(n=64–79 cells per group) and (j)(n=6 mice per group). Statistics were calculated with one-way ANOVA in (c,d)(****p<0.0001), two-way ANOVA in (f) and two-sided unpaired Student’s t-test (**p<0.01, ****p<0.0001) in (b,e,g,h and j) with Bonferroni correction. Exact P values and adjustments are provided in Source data. The error bars represent the mean ± s.e.m.

Article Snippet: Samples were stained with Rabbit anti-Red Fluorescence protein (RFP), Goat anti-mouse IgA (C10–3, BD Biosciences, used 1:500), Rat anti-mouse LYVE1 (223322, RD system, used 1:100), Rat anti-CD326 (G8.8, Biolegend, used 1:200), followed by Cy3 anti-goat Donkey (polyclonal, Jackson ImmunoResearch Lab, used 1:1000), AF647 anti-rat Goat (polyclonal, Jackson ImmunoResearch Lab., used 1:1000) and DAPI.

Techniques: Immunofluorescence, Flow Cytometry, Migration, Concentration Assay, Enzyme-linked Immunospot, Transduction, Plasmid Preparation, Injection, Irradiation

Journal: Nature immunology

Article Title: An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut resident plasma cells

doi: 10.1038/s41590-022-01413-w

Figure Lengend Snippet: a, Relative migration of lamina propria PCs from NIBR189 or vehicle (DMSO) treated mice and with the indicated concentration of GPR183L. b, Representative immunofluorescence of duodenum sections from mice treated twice with GPR183 inhibitor, NIBR189, or vehicle (DMSO).Scale bar is 50μm. c, Quantification of IgA+PCs frequency at certain distance from lymphatics (LYVE1+) showed as violin plot. Each dot represents the average of PCs frequency at the indicated range of n=7 mice per each treatment. d, Quantification of PCs distance from lymphatics upon GPR183 inhibition. Each dot represents a single PC. The results were pooled from two independent experiments (a)(n=3 mice pre group) and three independent experiments (b,c and d) (n=7 mice per group or n=62 cells (d)). Statistics were measured as two-sided unpaired Student’s t-test (***p<0.001,****p<0.0001) in (a,c and d). Exact P values and adjustments are provided in Source data. The error bars represent the mean ± s.e.m.

Article Snippet: Samples were stained with Rabbit anti-Red Fluorescence protein (RFP), Goat anti-mouse IgA (C10–3, BD Biosciences, used 1:500), Rat anti-mouse LYVE1 (223322, RD system, used 1:100), Rat anti-CD326 (G8.8, Biolegend, used 1:200), followed by Cy3 anti-goat Donkey (polyclonal, Jackson ImmunoResearch Lab, used 1:1000), AF647 anti-rat Goat (polyclonal, Jackson ImmunoResearch Lab., used 1:1000) and DAPI.

Techniques: Inhibition, Migration, Concentration Assay, Immunofluorescence

Journal: Nature immunology

Article Title: An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut resident plasma cells

doi: 10.1038/s41590-022-01413-w

Figure Lengend Snippet: a, Representative immunofluorescence of PCs (IgA+), lymphatics (LYVE1+) and IECs (EPCAM+) on duodenum sections from wild type mice. Scale bar is 100μm (left) and 50 μm (right). b, Representative immunofluorescence of PCs (IgA+), lymphatics (LYVE1+) and IECs (EPCAM+) in PCΔGpr183 and IECΔCh25h sections and compiled data (c,d). Scale bar is 100μm (left) and 50 μm (right). Each dot represents the average of PCs frequency at the indicated range of distance measured in a total of n=7 mice per each treatment. e, Representative immunofluorescence of PCs (IgA+), lymphatics (LYVE1+) and DAPI in duodenum sections of mice fasted for 12hrs, refed once with HCF and then harvested at 3 or 12 hrs post-HCF. Scale bar is 50 μm. f, Frequency of PCs at the indicated distance from lymphatics from mice in (e). Each dot represents the average of PCs frequency at the indicated range of distance measured in n=6 mice per each treatment. g, Representative IgA ELISPOT and number (h), area and intensity of IgA spots from lamina propria of fasted mice for 12 hours (I), refed with HCF and fasted again for 6 hours (II). The results were pooled from three independent experiments (a,b,c,d,e and f)(n=6–7 mice pre group) and 2 independent experiments (g and h)(n=5 mice per group or n=40–68 cells). Statistics were measured as two-sided unpaired Student’s t-test (*p<0.05, ****p<0.0001) with Bonferroni’s correction in (c and d), two-way ANOVA (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001) in (f) and one-way ANOVA in (h) with Bonferroni’s correction. Exact P values and adjustments are provided in Source data. The error bars represent the mean ± s.e.m.

Article Snippet: Samples were stained with Rabbit anti-Red Fluorescence protein (RFP), Goat anti-mouse IgA (C10–3, BD Biosciences, used 1:500), Rat anti-mouse LYVE1 (223322, RD system, used 1:100), Rat anti-CD326 (G8.8, Biolegend, used 1:200), followed by Cy3 anti-goat Donkey (polyclonal, Jackson ImmunoResearch Lab, used 1:1000), AF647 anti-rat Goat (polyclonal, Jackson ImmunoResearch Lab., used 1:1000) and DAPI.

Techniques: Immunofluorescence, Enzyme-linked Immunospot

Journal: Nature immunology

Article Title: An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut resident plasma cells

doi: 10.1038/s41590-022-01413-w

Figure Lengend Snippet: a, Representative immunofluorescence of duodenum sections from mice treated for 24 hrs with NF, HCF and VF. b, Quantification of PC frequency at the indicated distance from lymphatics (LYVE1+) in duodenum of mice treated as in (a). Each dot represents the average of PCs distance measured from n=6 mice per each treatment. c, Quantification of PCs distance from lymphatics in mice treated with the indicated food changes. Each dot represents a single PC. d, Representative IgA ELISPOT and compiled data from duodenum of untreated or fasted mice for 12 hours. e, Frequency of IgA+CD98hi cells analyzed by flow cytometry in LP of mice fasted for 12hrs, or untreated. The results were pooled from three experiments (a,b and c)(n=7 mice per group and n=50–145 cells (c)) and two independent experiments (d,e)(n=3–5 mice per group). Statistics were calculated with two-way ANOVA with Bonferroni’s correction (*p<0.05, **p<0.01,***p<0.001, ****p<0.0001) in (b and c) and two-sided unpaired Student’s t-test (**P<0.01,****P<0.0001) in (d and e). Exact P values are provided in Source data. The error bars represent the mean ± s.e.m.

Article Snippet: Samples were stained with Rabbit anti-Red Fluorescence protein (RFP), Goat anti-mouse IgA (C10–3, BD Biosciences, used 1:500), Rat anti-mouse LYVE1 (223322, RD system, used 1:100), Rat anti-CD326 (G8.8, Biolegend, used 1:200), followed by Cy3 anti-goat Donkey (polyclonal, Jackson ImmunoResearch Lab, used 1:1000), AF647 anti-rat Goat (polyclonal, Jackson ImmunoResearch Lab., used 1:1000) and DAPI.

Techniques: Derivative Assay, Immunofluorescence, Enzyme-linked Immunospot, Flow Cytometry