Journal: PLoS ONE

Article Title: Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

doi: 10.1371/journal.pone.0121550

Figure Lengend Snippet: ( A - I ) In situ hybridizations for Alcam on lumbar spinal cord sections from E16.5 ( A - C ), P0 ( D - F ), and P4 ( G - I ) wild-type mice. Alcam was strongly expressed by a subset of sensory and motor neurons at E16.5. Alcam was ubiquitously expressed in the spinal cord at P0 and P4.

Article Snippet: We used rabbit anti-parvalbumin (Pv) (Swant), rabbit anti-GFP (Molecular Probes), goat anti-human Alcam (R & D systems), goat anti-mouse Alcam (R & D systems), goat anti-Mcam/CD164 (R & D systems), mouse Anti-choline acetyltransferase/ChAT (Chemicon), goat anti-Islet1 (R & D systems), guinea pig anti-Foxp1 (kindly provided by Dr. Bennett G. Novitch, University of California, Los Angeles, Los Angeles, CA), rabbit anti-Lhx3 (kindly provided by Dr. Kamal Sharma, University of Chicago, Chicago, IL), and rabbit anti-Ocam antibodies [ ].

Techniques: In Situ

Journal: PLoS ONE

Article Title: Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

doi: 10.1371/journal.pone.0121550

Figure Lengend Snippet: ( A - I ) Transverse sections of the lumbar spinal cord and DRG at E16.5 ( A - C ) and P0 ( D - I ) were immunostained for Alcam, Pv, and ChAT. Alcam was strongly expressed by a subset of sensory neurons in the DRG ( A - C ). Some of the Alcam + sensory neurons were Pv + (arrows). ( D - F ) Alcam was also detected in sensory axons. Axons of Alcam + and Pv − cutaneous sensory neurons terminated in the dorsal horn, whereas axons of Alcam + and Pv + proprioceptive sensory neurons projected to the ventral horn. ( G - I ) A high power view of ventral spinal cord, showing that some of Alcam + and Pv + axons were in close proximity to Alcam + motor neurons.

Article Snippet: We used rabbit anti-parvalbumin (Pv) (Swant), rabbit anti-GFP (Molecular Probes), goat anti-human Alcam (R & D systems), goat anti-mouse Alcam (R & D systems), goat anti-Mcam/CD164 (R & D systems), mouse Anti-choline acetyltransferase/ChAT (Chemicon), goat anti-Islet1 (R & D systems), guinea pig anti-Foxp1 (kindly provided by Dr. Bennett G. Novitch, University of California, Los Angeles, Los Angeles, CA), rabbit anti-Lhx3 (kindly provided by Dr. Kamal Sharma, University of Chicago, Chicago, IL), and rabbit anti-Ocam antibodies [ ].

Techniques:

Journal: PLoS ONE

Article Title: Expression of the Immunoglobulin Superfamily Cell Adhesion Molecules in the Developing Spinal Cord and Dorsal Root Ganglion

doi: 10.1371/journal.pone.0121550

Figure Lengend Snippet: ( A - F ) Transverse sections of the lumbar spinal cord from E15.5 Olig2-Cre ; ccGFP embryos were immunostained for Alcam ( A , B ), Mcam ( C , D ), Ocam ( E , F ) and GFP ( B, D, and F ) expressions. All three IgSF molecules were expressed by a subset of motor neurons (arrows).

Article Snippet: We used rabbit anti-parvalbumin (Pv) (Swant), rabbit anti-GFP (Molecular Probes), goat anti-human Alcam (R & D systems), goat anti-mouse Alcam (R & D systems), goat anti-Mcam/CD164 (R & D systems), mouse Anti-choline acetyltransferase/ChAT (Chemicon), goat anti-Islet1 (R & D systems), guinea pig anti-Foxp1 (kindly provided by Dr. Bennett G. Novitch, University of California, Los Angeles, Los Angeles, CA), rabbit anti-Lhx3 (kindly provided by Dr. Kamal Sharma, University of Chicago, Chicago, IL), and rabbit anti-Ocam antibodies [ ].

Techniques:

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: Production and characterization of the ALCAM-blocking antibody I/F8-Fc. (A) Schematic representation of the homodimeric I/F8-Fc antibody, consisting of scFv IF/8 fused to the Fc portion of murine IgG 1 . (B) SDS-PAGE of I/F8-Fc under non-reducing (nr) and reducing (r) conditions. Expected molecular weight: 51 kDa under reducing conditions. (C) In FPLC I/F8-Fc elutes as a homodimer. (D,E) Surface plasmon resonance profiles of purified I/F8-Fc on micro sensor chips coated with (D) human and (E) murine ALCAM. (F,G) FACS analysis reveals I/F8-Fc binding to (F) human and (G) murine endothelial cell lines. (H–J) Analysis of I/F8-Fc specificity by FACS analysis performed on single cell suspensions generated from murine lungs of WT and ALCAM −/− mice. (H) FACS gating strategy for differentiating BECs from LECs. Single-cell suspensions from enzymatically digested lung tissue were stained for CD45, CD31, and podoplanin. BECs and LECs were identified, by gating on CD45 − CD31 + podoplanin − (BECs) or CD45 − CD31 + podoplanin + (LECs) cells. (I,J) I/F8-Fc stains BECs (CD45 − CD31 + podoplanin − ) and LECs (CD45 − CD31 + podoplanin + ) in lung single-cell suspension derived from (I) WT mice but not from (J) ALCAM −/− mice. Black lined histogram: I/F8-Fc, shaded histogram: KSF-Fc control. Representative data from one out of four similar experiments are shown in (F–J) .

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Blocking Assay, SDS Page, Molecular Weight, SPR Assay, Purification, Binding Assay, Generated, Staining, Suspension, Derivative Assay, Control

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: I/F8-Fc reduces (lymph)angiogenic processes and T cell activation in vitro . (A–E) Effects on in vitro (lymph)angiogenis. (A,B) A cell-free scratch was introduced into confluent monolayers of (A) human LECs or (B) HUVECs and the impact of I/F8-Fc or KSF-Fc control antibody on VEGF-A-induced scratch closure was analyzed after 24 and 12 h, respectively (C) Blocking ALCAM with I/F8-Fc reduced tube formation of human LECs. (D,E) A cell-free scratch was introduced into confluent monolayers of (D) murine MS-1 cell or (E) murine primary dermal LECs and the impact of I/F8-Fc on scratch closure was analyzed after 24 and 27 h, respectively. Data from 1 out of 3 to 4 similar experiments are shown in (A–E) . (F–I) Effects on in vitro T cell activation. WT or ALCAM −/− BM-DCs were pulsed with OVA peptide in presence of LPS and co-incubated with CD4 + OTII cells in presence of I/F8-Fc or KSF-Fc control antibody. (F) FACS analysis demonstrating ALCAM and CD6 expression in BM-DCs and OTII cells, respectively. (G,H) Impact of I/F8-Fc treatment on T cell proliferation. (G) Representative FACS plots showing CFSE-dilution, as a readout of T cell proliferation. (H) Quantitation of proliferating cells. (I) T cell-mediated IFN-γ production was quantified in the cell culture supernatants. Data from 1 out of 4 similar experiments ( n = 6 replicates) are shown in F-I. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Activation Assay, In Vitro, Control, Blocking Assay, Incubation, Expressing, Quantitation Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: I/F8-Fc treatment reduces developmental lymphangiogenesis in the diaphragm to a similar degree as ALCAM deficiency. (A) Schematic representation of the diaphragm and of the area analyzed by LYVE-1 immunofluorescence in D-J. TW: thoracic wall; CT: central tendon. (B) ALCAM expression on diaphragmatic P5 BECs (CD45 − CD31 + podoplanin − ) and LECs (CD45 − CD31 + podoplanin + ) was confirmed by FACS. Data from 1 out of 2 similar experiments are shown. Specific stainings are shown as black lined and isotype control stainings as gray tinted histograms. (C) Schematic presentation of the antibody treatment protocol. Pregnant mice received I/F8-Fc or KSF-Fc antibody i.p. (300 μg) on day E15.5 and E17.5 (indicated by an asterisk). In addition, newborn mice received antibody i.p. (30 μg) on day P1 and P3 (indicated by a circle). (D–H) On P5 the diaphragm was collected and lymphatic vessels were visualized by LYVE-1 staining. (D,E) Representative stainings from diaphragms of KSF-Fc- or I/F8-Fc-treated animals. (F–H) Image-based morphometric analysis of (F) the LYVE-1 + area, (G) the number of branch points and (H) the total vessel length in the diaphragm of I/F8-Fc or KSF-Fc control-treated pups. Each dot represents the measurement made in one animal ( n = 10). Data from 1 out of 3 similar experiments are shown in F-H. (I,J) Image-based morphometric analysis of (I) the number of branch points and (J) the total vessel length in the P5 diaphragm of WT and ALCAM −/− mice. Data from 1 experiment ( n = 6 or 9 mice) are shown in G-H. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Immunofluorescence, Expressing, Control, Staining

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces DC transmigration and crawling on lymphatic endothelium in vitro . (A,B) imLECs were seeded on the lower surface of transwell inserts and grown to confluence. Transmigration of BM-derived WT or ALCAM –/– BM-DCs across imLEC monolayers was investigated in presence of either KSF-Fc (control) or I/F8-Fc antibody. (A) Absolute numbers and (B) % of transmigrated DCs from the input. Each dot represents the value obtained from one transwell. Pooled data from 3 similar experiments (involving 3 transwells per condition per experiment) are shown in (A,B) . (C) DCs isolated from either WT or ALCAM −/− mice were treated with I/F8-Fc or KSF-Fc antibody, followed by analysis of crawling speed on ALCAM-expressing imLEC monolayers. Each dot represents one DC. Pooled data from 2 to 3 similar experiments are shown. KSF-Fc: control antibody. I/F8-Fc: anti-ALCAM.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Transmigration Assay, In Vitro, Derivative Assay, Control, Isolation, Expressing

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces corneal allograft rejection. (A) Schematic description of the corneal allograft rejection model. On day −14 three sutures were placed into the corneas of recipient BALB/c mice. On day 0, sutures were removed and the donor corneas from C57BL/6 mice were transplanted. Mice received I/F8-Fc or KSF-Fc control antibody i.p. (150 μg) on day −1, 2, 5, 8, and 12 (indicated by an asterisk). Additionally, mice were treated by topical application of antibody (3 μg) twice a day for 14 days (day 3–14; gray bar). Transplant survival was monitored over 8 weeks (day 0–56). Transplants were considered rejected when the cornea lost its transparency. (B) Representative images of a surviving and a rejected cornea. The graft border is indicated by an asterisk. (C) Impact of antibody treatment on corneal allograft survival. NR group: normal risk group, consisting of mice that received a cornea transplant without the previous induction of suture-induced inflammation. Statistical analysis: I/F8-Fc vs. KSF-Fc: p = 0.024; I/F8-Fc vs. NR: p = 0.77; KSF-Fc vs. NR: p = 0.019. (D–F) FACS analysis was performed on eye-draining cervical LNs of all mice at the end of the study. Percentage of (D) CD45 + CD3 + T cells, (E) CD45 + CD11c + DCs (F) and CD45 + CD4 + Foxp3 + Tregs in cervical LNs. Pooled data from 2 experiments with a total of 10 mice /group are shown.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade results in accumulation of corneal DCs. (A) Schematic description of the suture model. Three sutures were placed into the corneas. Mice received I/F8-Fc or KSF-Fc control antibody i.p. (150 μg) on day 2, 5, 8, and 12 (indicated by an asterisk) as well as antibody-containing eye drops (3 μg) twice a day for 14 days (gray bar). Corneas were analyzed by whole-mount immunofluorescence on day 14 ( n = 5/group). (B–G) Representative immunofluorescence images and quantifications of the percentage of corneal area covered with (B,C) LYVE-1 + lymphatic vessels (LVs), (D,E) CD31 + blood vessels (BVs), scale bars: 500 μm, and (F,G) CD11c + DCs, scale bars: 100 μm. Images on the left in (F) show CD11c + cells (green) detected by immunofluorescence. Images on right show corresponding cells (red) identified by gray-image threshold analysis, which was used for quantification (G) . (H) Schematic representation of the suture/allograft model: three sutures were placed into the corneas and animals were treated as in (A) . On day 14 corneal transplantation was performed. Five days after transplantation (day 19), corneas were analyzed by whole-mount immunofluorescence ( n = 10/group). (I–L) Representative images and quantifications of the percentage of corneal area covered with (I,J) CD11c + DCs and (K,L) CD3 + T cells. DC and T cell quantification in (I–L) was performed as in (F,G) . Images on the left in (I,K) depict DC and T cells as seen in immunofluorescence; images on the right (in red) as identified by gray-image threshold analysis. Scale bars: 100 μm.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Control, Eye Drops, Immunofluorescence, Transplantation Assay

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM blockade reduces DC emigration from human skin. Human skin sections were stained for (A) LYVE-1 and (B) MHCII, in combination with either an ALCAM-specific or the corresponding isotype control antibody. ALCAM reactivity was observed in (A) all LYVE-1 + vessels and (B) in some MHCII + cells. Scale bars: 50 μm. (C) Human skin punch biopsies were incubated in culture medium and the presence of ALCAM on emigrated CD86 + MHCII hi cells was analyzed after 24 h. Data from 1 representative of a total of 3 experiments are shown. (D) Human skin DC emigration experiment. Punch biopsies of human skin were incubated in culture medium in presence of either I/F8-Fc or of KSF-Fc control antibody, and cells emigrated into the culture medium were analyzed by FACS 24 h later. In (C) representative FACS plots show the gating scheme. Migratory DCs were identified as CD86 + MHCII hi cells and were quantified with the help of counting beads. Each dot represents one biopsy ( n = 10 per condition).

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Staining, Control, Incubation

Journal: Frontiers in Immunology

Article Title: ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions

doi: 10.3389/fimmu.2019.00759

Figure Lengend Snippet: ALCAM is expressed in pathologic blood and lymphatic vessels in neovascularized human corneas and in corneal DCs. Serial sections of inflamed human corneas obtained from high risk transplant patients were stained for (A) ALCAM and LYVE-1 and (B) ALCAM and MHC-II. Magnifications of the boxed areas are shown in the center part of the Figure. LV, Lymphatic vessel; BV, Blood vessel, E, epithelium. Arrows indicate specific staining. Scale bar: 20 μm. Representative images from 3 stained neovascularized corneas are shown.

Article Snippet: ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen).

Techniques: Staining

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Regulation of fibroblast lipid storage and myofibroblast phenotypes during alveolar septation in mice.

doi: 10.1152/ajplung.00144.2014

Figure Lengend Snippet: Fig. 3. FACS analysis of progenitor markers in fibroblasts (LF) with different levels of pdgfr gene expression. LF isolated from PDGFR-GFP mice at P8 were stained for the antigens shown on the y-axes: LTR, neutral lipid binding LTR (A); ADRP, adipocyte differentiation related protein, perilipin-2 (B); -SMA, -smooth muscle actin, acta2; (C); Sca1, stem cell antigen-1 (D); CD34, podocalyxin (E); and CD166, activated leukocyte adhesion molecule (ALCAM), activated-leukocyte cell adhesion molecule (F); the CD45 cell populations were selected as shown in Fig. 2 and expressed as a percentage of all CD45 LF within a particular population based on the intensity of GFP fluorescence (GFP, GFPlow, GFPhigh). **P 0.01 and *P 0.05, mean SE, n 6 mice, 2-way ANOVA, Student-Newman-Keuls post hoc test comparing GFPlow or GFPhigh LF with GFP. †P 0.01 comparing GFPlow with GFPhigh.

Article Snippet: Primary antibodies were mouse monoclonal anti- -smooth muscle actin ( -SMA), clone IH8 (MA5-15805; Thermo Scientific, Rockford, IL); mouse monoclonal clone IA4, anti- -SMA-PE (R&D Systems, Minneapolis, MN); mouse monoclonal -SMA clone IA4-FITC (Sigma-Aldrich, St. Louis, MO); goat polyclonal anti-mouse CD166 (R&D Systems); mouse monoclonal IgG1 anti-Dlk1, clone 3A10 (MA5-15915; Thermo Scientific); goat anti-MRTF-A (sc-21558, Western 1:500 dilution; Santa Cruz Biotech, San Jose, CA); anti-Sox9 (rabbit polyclonal, AB5535, Western 1:2,000 dilution; Millipore, Temecula, CA); and mouse monoclonal anti- -tubulin (clone TUB2.1; Sigma-Aldrich).

Techniques: Gene Expression, Isolation, Staining, Binding Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Regulation of fibroblast lipid storage and myofibroblast phenotypes during alveolar septation in mice.

doi: 10.1152/ajplung.00144.2014

Figure Lengend Snippet: Fig. 6. Targeted pdgfr gene deletion alters the abundance of Dlk1, Sox9, and Tcf21. A–D: FACS analysis of LF isolated at P8 from 5 mice bearing the transgelin-mediated (TG-Cre) conditional deletion of loxP-flanked pdgfr (PDGFRF/F) or 5 littermate controls, gating on CD45 cells. A: %Dlk1, LTR, or Dlk1,Sca1 gating on CD45 (left); or %LTR within CD45 (axis on right). B: Dlk1 LF within the CD45, CD34 population (axis on left); %CD45, CD34 relative to all CD45 LF (axis on right). C: %CD166 (axis on left) or both CD166, Dlk1 (axis on right) in the CD45 population. *P 0.05, n 5, mean SE, t-test, paired variables. D: %CD45 LF containing -SMA in control (open bars) or TGCre/;PDGFRF/F (solid bars, axis on right). %Sox9, CD45, -SMA subpopulation, expressed relative to all -SMA LF (axis on left). E: qRT-PCR analysis of periostin (Postn), periostin-lacking exon 21 (Postn 20–21), tcf21, and ppar in PDGFR-deleted and controls within the same litter, n 5 litters. *P 0.05 and **P 0.01, mean SE, t-test, paired variables comparing PDGFRF/F LF with those from their respective littermate controls. F: qRT-PCR using a probe that is not influenced by splicing dlk1-T (total), the A and C splice variants (dlk1-A and dlk1-C, respectively) comparing control and TGCre/;PDGFRF/F mice (mean SE, n 3 for each group); *P 0.05, n 3. dlk1-T was normalized to 2-microglobulin mRNA, whereas the dlk1-A and -C splice variants were normalized to dlk1-T for each respective sample.

Article Snippet: Primary antibodies were mouse monoclonal anti- -smooth muscle actin ( -SMA), clone IH8 (MA5-15805; Thermo Scientific, Rockford, IL); mouse monoclonal clone IA4, anti- -SMA-PE (R&D Systems, Minneapolis, MN); mouse monoclonal -SMA clone IA4-FITC (Sigma-Aldrich, St. Louis, MO); goat polyclonal anti-mouse CD166 (R&D Systems); mouse monoclonal IgG1 anti-Dlk1, clone 3A10 (MA5-15915; Thermo Scientific); goat anti-MRTF-A (sc-21558, Western 1:500 dilution; Santa Cruz Biotech, San Jose, CA); anti-Sox9 (rabbit polyclonal, AB5535, Western 1:2,000 dilution; Millipore, Temecula, CA); and mouse monoclonal anti- -tubulin (clone TUB2.1; Sigma-Aldrich).

Techniques: Isolation, Control, Quantitative RT-PCR

Journal: Genes

Article Title: Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources

doi: 10.3390/genes12030366

Figure Lengend Snippet: List of primary antibodies used for flow cytometry.

Article Snippet: CD166 , rabbit IgG , polyclonal , purified 1 , Bioss.

Techniques: Cytometry, Purification