Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Summary of Primary Antibodies Used in These Studies

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Generated

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Human neuronal precursors were mitotic and E-NCAM+. First-passage cells were acutely dissociated, plated, and processed for immunocytochemistry 48 hr later. A: First-passage HNPs displayed IR for E-NCAM (A, red) and β-III tubulin (A″, green). DAPI staining of nuclei (A′, blue) shows that only a subset of the cells expresses neuronal markers. β-III Tubulin+ and E-NCAM+ cells (B,C, green) incorporated BrdU (B,C, red), indicating that they were mitotic (arrowheads indicate double-labeled cells). β-III Tubulin+ cells (D, red) coexpressed another neuronal-specific protein, MAP2 (D′, green). β-III Tubulin+ cells (F, green, and G, red) did not coexpress the glial markers A2B5 (F, red) or GFAP (G, green). Staining was performed on two or three dishes of cells from four different isolations. E shows human NRP antigenic profiles.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Immunocytochemistry, Staining, Labeling

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Immunostaining Profiles (%) of Human Fetal Brain Culture *

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Immunostaining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Immunostaining Profiles (%) of Differentiated E-NCAM + HNPs *

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Immunostaining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: HNPs do not coexpress nestin. Acutely dissociated human neural cells were grown for 5 days in culture, pulsed with BrdU for 16 hr (E,F), fixed, and processed for immunocytochemistry to detect the expression of nestin (A,B,D,F, red, and C,E, green) and GFAP (B, green), E-NCAM (C, red), β-III tubulin (D,F green), and BrdU (E, red, and F, blue). DAPI staining (A, blue) was used to identify all cells. Large numbers of nestin+ cells were present in culture and made up about 70% of the total cell population (A). About 15% of the nestin+ cells coexpressed GFAP IR (B, arrowheads), although a few GFAP+ cells did not express nestin (B, arrows). Five percent of the E-NCAM+ cells were nestin+ (C, arrowheads), but none of the β-III tubulin+ cells was nestin+ (D). BrdU labeling identified dividing cells (E,F) and showed that BrdU incorporation was seen in both nestin+ (E,F, arrowhead) and nestin− (E,F, arrow) cells. Triple labeling showed that some of the nestin−, BrdU+ cells (F, arrowheads) were β-III tubulin+ (green). Only 5% of the β-III tubulin+ cells incorporated BrdU, and postmitotic BrdU−, nestin−, β-III tubulin+ cells were also seen (F, arrow, green). Staining was performed on two or three dishes of cells from four different isolations.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Immunocytochemistry, Expressing, Staining, Labeling, BrdU Incorporation Assay

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed synaptophysin and neurotransmitter-synthesizing enzymes. Cells were maintained under differentiating conditions for 14 DIC and processed for immunocyto-chemistry for E-NCAM (A), synaptophysin (A′), glutamate (B), glycine (C), TH (D), and ChAT (E). A single cell exhibiting synaptophysin IR is shown (A′), and expression colocalizes with that of E-NCAM (A). Subsets of cells express IR for glutamate (B, arrowhead), glycine (C, arrowhead), TH (D, arrowhead), and ChAT (E, arrowhead), whereas other cells are negative (B–E, arrows). Staining was performed on two or three dishes of cells from two different isolations.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Expressing, Staining

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: E-NCAM+ HNPs immunoidentified for patch-clamp and fura-2 recordings. Shown are mixed human cell cultures from undifferentiated conditions (A) or following 14 days under differentiating conditions (B), visualized using Hoffman optics. The cells were live stained for E-NCAM (red in A′, B′). A composite Hoffman-fluorescence image (A″, B″) shows the red E-NCAM+ HNPs from which whole-cell currents were recorded (arrows). Identical methods were used to identify E-NCAM+ cells used for Ca2+ imaging experiments.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Patch Clamp, Staining, Fluorescence, Imaging

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed voltage-gated Na+ and K+ channels and fired action potentials. A: Whole-cell voltage-clamp recordings were made from E-NCAM+ HNPs in acutely passaged conditions (A1,A2) or following differentiation (A3). Cells were held at −100 mV and stepped to test voltages between −80 and 80 mV in 10 mV increments. Both of the acute HNPs expressed outward K+ currents (A1,A2), but only one exhibited a small inward Na+ current (A2). A differentiated HNP expressed both outward K+ and inward Na+ currents (A3). B1–3: Peak outward K+ currents (triangles) and peak inward Na+ currents (circles) were plotted against the command voltage for the cells represented in A. C: HNPs under acutely passaged conditions failed to fire action potentials (C1,C2); in contrast, a differentiated HNP fired an action potential when stimulated with depolarizing current injections (C3). Inset in C3 shows the action potential overshoot on a longer time scale.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques:

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Differentiating HNPs expressed functional neurotransmitter receptors. A,B: Ratiometric imaging of acute and differentiating E-NCAM+ HNP cells. Ratio of fura-2 emission (I340/I380, left y axis) with approximate Ca2+ concentrations ([Ca2+]i, right y axis). Arrow-heads and labels show application of neurotransmitters. All substances were applied at 500 μM, except for K50, which was 50 mM K+ HR. A: An acutely passaged HNP responded only to acetylcholine (ACh), with small magnitude. B: A differentiated neuron responded to GABA, glutamate (E), glycine (G), elevated K+ (K50), and ACh. C: Fraction of cells responding to neurotransmitters is shown for acutely passaged cells (open bars, n = 17) and differentiated cells [shaded bars, n = 70, except for norepinephrine (NE), for which n = 43]. Acute HNPs did not respond to GABA, glycine, dopamine (DA), NE, or ascorbic acid (AA) control. Differentiated neurons responded to all substances with higher frequencies, but only ACh and NE were significant (asterisks). D: The distribution of response amplitudes is shown by the box plot of acute (left box, open circles) and differentiating cells (Diff; right box, gray circles). Squares represent distribution mean, box represents the 75th percentiles, error bars represent the 95th percentiles, and circles plot the individual response amplitudes. Experiments were performed on a total of six dishes of cells from three different isolations.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Functional Assay, Imaging, Control

Journal:

Article Title: Identification and Characterization of Neuronal Precursors and Their Progeny From Human Fetal Tissue

doi:

Figure Lengend Snippet: Subset of E-NCAM- and β-III tubulin-immunoreactive HNPs coexpresses α-tubulin promoter-driven GFP. Tα1:GFP constructs were transfected into day 2 cultures of fetal neural cells. GFP expression (A,A′, A″, B,C,D, green) was seen in cells with neuronal morphology as early as 24 hr after transfection. Plates were fixed and processed for E-NCAM IR (B, inset, B′, red), β-III tubulin (C, red), and GFAP (D, red). The inset shows lack of colocalization for Tα1:GFP (green) and E-NCAM (red). We show one Tα1:GFP+ cell that was E-NCAM+ (B,B′, arrowhead) and one that was β;-III tubulin+ (C, arrowhead). None of the Tα1:GFP-expressing cells was GFAP+ (arrows in C and D show lack of colocalization). These experiments were performed on two or three dishes of cells from three different isolations.

Article Snippet: All secondary antibodies were purchased from either Jackson Immunoresearch Laboratories or Southern Biotechnology Associates Inc. (Birmingham, AL). table ft1 table-wrap mode="anchored" t5 TABLE I caption a7 Antigen/antibody Type Species Clonality Dilution Source E-NCAM IgM Hybridoma Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB), University of Iowa, Iowa City, IA A2B5 IgM Hybridoma Monoclonal 1:1 ATCC, Rockville, MD β-III Tubulin IgG2b Mouse Monoclonal 1:100 Sigma, St. Louis, MO MAP2 IgG Mouse Monoclonal 1:500 Sigma Human nestin IgG Rabbit Polyclonal 1:400 Generated by C. Messam at the NIH-NINDS, Bethseda, MD RT-97 IgG1 Mouse Monoclonal 1:1 Developmental Hybridoma Studies Bank (DHSB) Synaptophysin IgG Mouse Monoclonal 1:100 Sigma NF-150 IgG Mouse Monoclonal 1:500 Sigma ChAT IgG Goat Polyclonal 1:100 Chemicon, Temecula, CA TH IgG Rabbit Polyclonal 1:500 Chemicon Glutamate IgG Rabbit Polyclonal 1:100 Signature Immunologicals, Salt Lake City, UT Glycine IgG Rabbit Polyclonal 1:100 Signature Immunologicals GFAP IgG Rabbit Polyclonal 1:500 Dako, Carpinteria, CA O4 IgM Mouse Monoclonal 1:25 Gift of Dr. M. Mayer-Proschel, University of Rochester, Rochester, NY Gal C IgG Mouse Monoclonal 1:5 Gift of Dr. M. Mayer-Proschel Open in a separate window Summary of Primary Antibodies Used in These Studies

Techniques: Construct, Transfection, Expressing

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Representative immunofluorescence of E-cadherin (Red) and DAPI (Blue) in mice lungs showing knockdown of E-cadherin in adeno-Cre (Ad5CMVCre-eGFP) intratracheally instilled group compared to adeno-Ctrl (Ad-5CMVeGFP) after a 10 days (scale bar of 50 µm) and b 1 month (scale bar of 100 µm) with 10× objective. c Decreased fluorescence intensity of E-cadherin in the mice lungs intratracheally instilled with adeno-Cre compared to adeno-Ctrl. Increased terminal airway/airspace enlargement as observed by d H & E staining (representative image) of mice lung parenchyma at 5× (scale bar of 500 µm), and e increased mean linear intercepts (Lm). Lung function was analyzed, where knockdown of E-cadherin with 1-month instillations of adeno-Cre compared to adeno-Ctrl in Cdh1 flox mice ( Cdh1 fl/fl ) caused f total lung capacity was increased, g residual volume was not altered, and h compliance was increased. Immunofluorescence of E-cadherin (Red) and DAPI (Blue) in mice lungs showing knockdown of E-cadherin among adeno-Cre intratracheal instilled group compared to adeno-Ctrl in Cdh1 fl/fl mice after 2-months and 3-months at 10× (scale bar of 100 µm) as observed in i representative immunofluorescence images (Left panel—2 months of instillations, Right panel—3 months of instillations) and j fluorescence intensity of E-cadherin (Top panel—2 months of instillations, Bottom panel—3 months of instillations). Increased terminal airway/airspace enlargement as observed in k H & E staining at 5× (scale bar of 500 µm) l increased Lm in adeno-Cre as compared to adeno-Ctrl instilled Cdh1 fl/fl mice (Left panel—2 months of instillations, Right panel – 3 months of instillations). Increased m total lung capacity, n residual volume, and o compliance in E-cadherin knockdown due to 2-months (left panel) and 3-months (right panel) instillations of adeno-Cre in Cdh1 fl/fl mice compared to adeno-Ctrl. Data is expressed as median bars and generated from 5 to 11 mice per group. Kruskal-Walli’s test followed by Dunn’s multiple comparison test was performed to compare the lung function tests (total lung capacity, residual volume, and compliance), whereas the Mann-Whitney test was performed to assess fluorescence intensity of E-cadherin and Lm. P < 0.05 were considered statistically significant. NI: Not instilled Cdh1 fl/fl mice.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Immunofluorescence, Knockdown, Fluorescence, Staining, Generated, Comparison, MANN-WHITNEY

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: To knock down E-cadherin in the AT1 cells of mice lungs, Cdh1 fl/fl -Ager Cre mice were fed a tamoxifen diet (TAM) for 1 month. These were compared to Cdh1 fl/fl -Ager Cre mice receiving a normal chow diet (ND) and Cdh1 fl/fl -Ager WT receiving TAM control mice. a Total lung capacity, b compliance, and c residual volume were decreased in E-cadherin knockdown in AT1 cells of mice lung. No difference in lung histology was observed by d H & E staining (representative image at 5X with a scale bar of 500 µm) and e quantified mean linear intercepts (Lm) after E-cadherin knockdown in AT1 cells. To knock down E-cadherin in the AT2 cells of mice lungs, Cdh1 fl/fl -Spc Cre mice were fed TAM for 1 month. These were compared to Cdh1 fl/fl -Spc Cre , mice receiving ND. f Total lung capacity was increased, g compliance was increased, and h without significant changes in residual volume in E-cadherin knockdown in AT2 cells of mice lungs. Lung histology shows airspace enlargement as observed by i H & E staining (representative image at 5× with a scale bar of 500 µm) and j an increase in Lm after E-cadherin knockdown in AT2 cells. Data is expressed as median bars and generated from 5 to 15 mice per group. Kruskal-Walli’s test followed by Dunn’s multiple comparison test was performed to assess total lung capacity, compliance, and residual volume in E-cadherin knockdown in AT1 cells. Mann-Whitney test was performed to assess Lm of AT1 and AT2, and lung function tests (total lung capacity, compliance, and residual volume) in E-cadherin knockdown in AT2 cells. Data is generated from 5 to 14 mice per group. P < 0.05 were considered statistically significant.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Knockdown, Control, Staining, Generated, Comparison, MANN-WHITNEY

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Regeneration of epithelium was assessed by BrdU staining. (a) Representative image at 10X (scale bar of 50 µm) of Cdh1 fl/fl mice instilled with adeno-Cre recombinase (Ad5CMVCre-eGFP) to knockdown E-cadherin for 3 months shows decreases in BrdU as compared to Cdh1 fl/fl instilled with adeno-Ctrl (Ad-5CMVeGFP). Decreases in the fluorescence intensity of b E-cadherin and c BrdU in Cdh1 fl/fl mice instilled with adeno-Cre as compared to Cdh1 fl/fl mice instilled with adeno-Ctrl. Data is generated from eight mice. To knock down E-cadherin in the AT2 cells of mice lungs, Cdh1 fl/fl -Spc Cre mice were fed tamoxifen (TAM) for 30 days. These were compared to Cdh1 fl/fl -Spc Cre mice receiving a normal chow diet (ND). In vivo knockdown of E-cadherin in Cdh1 fl/fl -Spc Cre mice show decreases in BrdU expression as observed in the d representative images at 10X (scale bar of 25 µm), with decreases in the intensity of e E-cadherin and f BrdU in AT2 cells. Data is generated from five mice. Undifferentiated normal basal epithelial cells were transfected with Ad-GFP-U6-h-CDH1-shRNA to knock down E-cadherin, and undifferentiated COPD cells were transfected with Ad-GFP-U6-h-CDH1 to overexpress E-cadherin. We compared to respective undifferentiated Normal/COPD with Ad-GFP. g Representative image at 40× (scale bar of 25 µm) and h quantification showing decreased BrdU intensity in the normal epithelium with E-cadherin knockdown (Normal + shCDH1) and COPD at baseline (COPD + GFP) as compared to Normal + GFP. Also, COPD with overexpressed E-cadherin (COPD + CDH1) demonstrates increased BrdU intensity compared to COPD + GFP. Data are expressed as median bars and generated from three inserts from two donors.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: BrdU Staining, Knockdown, Fluorescence, Generated, In Vivo, Expressing, Transfection, shRNA

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Immunofluorescence at 40× (scale bar of 50 µm) of COPD human bronchial epithelial cells differentiated at week one to three of air–liquid interface (ALI) show decreased expression of E-cadherin and β-tubulin (ciliated cells marker) expression, increase expression of MUC5AC (goblet cell marker), without any changes in Cytokeratin 14 (Basal cell marker), as compared to non-diseased human bronchial epithelial (normal) cells.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Immunofluorescence, Expressing, Marker

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: To knock down E-cadherin in the ciliated cells of mice lungs, Cdh1 fl/fl- Foxj1 Cre Het mice were fed tamoxifen (TAM) for 30 days. These were compared to Cdh1 fl/fl- Foxj1 Cre Het mice receiving a normal chow diet (ND) and Cdh1 fl/fl- Foxj1 Cre WT receiving TAM control mice. No change in a total lung capacity, b compliance, and c residual volume was observed in the ciliated cells with E-cadherin knockdown of mice lungs. Also, no changes were observed in d H & E staining (representative image at 5X with a scale bar of 500 µm), and e quantified mean linear intercepts (Lm) after E-cadherin knockdown in ciliated cells. f Increased airway hyperreactivity (AHR) in ciliated cells with E-cadherin knockdown. Data is expressed as median bars and representative of 5 to 9 mice. To knock down E-cadherin in the club cells of mice lungs, Cdh1 fl/fl- Scbg1a1 Cre mice were fed a TAM for 30 days. These were compared to Cdh1 fl/fl- Scbg1a1 Cre mice receiving an ND. No difference in g airway reactivity, and histology as observed in h H & E staining (representative image), and i MLI among Cdh1 fl/fl- Scbg1a1 Cre receiving a TAM and an ND. Data is expressed as median bars and generated from 5 to 9 mice. Kruskal-Wallis test followed by Dunn’s multiple comparison test was performed for total lung capacity, compliance, residual volume, and AHR in E-cadherin knock down in ciliated cells. Mann-Whitney test was performed for airway reactivity in E-cadherin knockdown in club cells and MLI values. P < 0.05 were considered statistically significant.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Knockdown, Control, Staining, Generated, Comparison, MANN-WHITNEY

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: To knock down E-cadherin in airways, mice tracheal epithelial cells (mTECs) from Cdh1 fl/fl mice cultured at the air-liquid interface (ALI) were transfected with Ad5CMVCre-eGFP (Cre) at 2 × 10 9 pfu and these were compared to Ad-5CMVeGFP (Ctrl). mTECs transfected with Cre show reduction in E-cadherin as assessed by a mRNA expression of Cdh1 (encodes for E-cadherin), and b western blotting (representative image – left panel, and quantification – right panel). c Epithelial resistance was decreased, and d cellular velocity was increased in mTECs with E-cadherin knockdown. Data is expressed as median bars and generated of cells derived from 12 mice, 4 to 12 inserts. Normal human bronchial epithelial cells at ALI (normal controls) were transfected with Ad-GFP-U6-h-CFL1-shRNA (shCDH1) at 1.5 × 10 9 pfu to knock down E-cadherin and these were compared to control adenovirus (Ad-GFP-U6-shRNA, GFP). Normal control cells transfected with shCDH1 show reduced e mRNA of CDH1 (encodes for E-cadherin) and f protein expression of E-cadherin (representative blot—left panel, and quantified blot—right panel). Assessment of the epithelial barrier function indicates that g monolayer resistance was decreased, h a trend towards increased permeability, and i cellular velocity was increased in control cells with knockdown of E-cadherin. Data is expressed as median bars and representative of 5 to 10 inserts per condition. Mann-Whitney test was performed and P < 0.05 was considered statistically significant.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Knockdown, Cell Culture, Transfection, Expressing, Western Blot, Generated, Derivative Assay, shRNA, Control, Permeability, MANN-WHITNEY

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: COPD cells at 4 to 6 weeks ALI were transfected Ad-GFP-U6-h-CDH1 (CDH1) to overexpress E-cadherin or Ad-GFP (GFP) as control at 2 × 10 9 pfu. COPD cells transfected with Ad-CDH1 result in a increased epithelial resistance, b reduced cellular velocity of COPD cells, and c increased mRNA expression of CDH1 . Overexpression of E-cadherin in COPD cells d – g increased mRNA expression of claudins— CLDN1 , CLDN3 , CLDN7 , and CLDN8 , h decreased mRNA expression of CLDN10, i increased mRNA expression of occludin (OCLN), j increased mRNA expression of tight junction protein 1 (TJP1), and k TJP2 was not altered. Data is expressed as median bars and generated from 4 to 12 transwells per condition from two donors. To induce the over-expression of E-cadherin in Cdh1 knock-in mice, mice tracheal epithelial cells (mTECs) were transfected with adeno-Cre (Ad5CMVCre-eGFP) at 2 × 10 9 pfu and were exposed to CS for 10 days. Overexpression of E-cadherin in mTECs protects against CS-induced epithelial functional phenotypes by l decreasing monolayer permeability, m decreasing the cellular velocity, and n protecting Cdh1 mRNA downregulation due to CS exposure. Data for l – m involves three to six transwells per condition. COPD cells treated with CDDO-Me restore epithelial function by o improving the epithelial resistance, p decreasing the cellular velocity, and q increasing the CDH1 mRNA expression of COPD cells as compared to age and gender-matched non-diseased epithelium (normal controls). Similarly, cigarette smoke (CS) exposed to healthy normal cells treated with CDDO-Me r restores epithelial resistance, s decreases cellular velocity, and t protects against CDH1 mRNA downregulation due to CS exposure. (Data for o – t is generated from 3 to 6 transwells from 2 donors). For the panels the same data for normal are used for a – k , and o – t in the figure. Data are expressed as median bars. Kruskal-Wallis test, followed by Dunn’s multiple comparison test was performed . P < 0.05 were considered statistically significant.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Transfection, Control, Expressing, Over Expression, Generated, Knock-In, Functional Assay, Permeability, Comparison

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: No difference in a total lung capacity, b residual volume, and c compliance among mice overexpressing E-cadherin in AT2 cells ( Cdh1 Oe -Spc Cre ) instilled with elastase as compared to Cdh1 Oe -Spc Cre with PBS and wild-type (Wt) instilled with elastase. d H&E staining (representative image) and e quantitative MLI showed a reduction in airspace enlargement as compared to Cdh1 Oe -Spc Cre instilled with PBS and Wt instilled with elastase. Data are expressed as median bars and generated from 5 to 14 mice. Kruskal-Wallis test, followed by Dunn’s multiple comparison test was performed . P < 0.05 were considered statistically significant.

Article Snippet: The primary antibody used in this study were Recombinant Anti-Prosurfactant Protein C antibody [EPR19839] (dilution of 1:100, ab211326, Abcam, MA, USA), Anti-BrdU antibody [IIB5] (5-Bromo-2’-deoxyuridine, Thymidine analog, dilution of 1:100, ab8152, Abcam, MA, USA), β-tubulin (D2N5G) Rabbit mAb #15115 (dilution of 1:200, Cell Signaling Technology, MA, USA), Cytokeratin 14 (LL001) (dilution of 1:100, sc-53253, Santa Cruz Biotechnology, Inc., TX, USA), E-cadherin (24E10) Rabbit mAb #3195 (dilution of 1:200, Cell Signaling Technology, MA, USA), E-cadherin (4A2) Mouse mAb #14472 (dilution of 1:100, Cell Signaling Technology, MA, USA), and Recombinant Anti-Mucin 5AC antibody [45MI] (dilution of 1:50, ab3649, Abcam, MA, USA), diluted in blocking solution, was incubated on the tissue slides overnight at 4 °C.

Techniques: Staining, Generated, Comparison