Journal: The Journal of Cell Biology

Article Title: Effects of Forced Expression of an NH 2 -terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

doi:

Figure Lengend Snippet: Forced expression of ΔN89β-catenin has no discernible effects on epithelial cell differentiation. Frozen sections were prepared from PLP-fixed jejunums of 6-mo-old chimeric-transgenic animals. ( A ) A polyclonal villus stained with biotin-conjugated Dolichos biflorus agglutinin ( DBA ), Cy3-conjugated avidin, rabbit anti–β-gal, and FITC-conjugated donkey anti–rabbit Ig. Glycoconjugates containing GalNAcα3GalNAc and GalNAcα3Gal recognized by DBA appear yellow-orange; β-gal appears green. The polarity and differentiation of enterocytes appears to be unaffected, as judged by the distribution of these glycoconjugates in apical membranes and supranuclear Golgi apparatus ( closed arrow ). Similarly, based on their reaction with DBA, the number and differentiation of goblet cells ( open arrows ) is equivalent in the 129/Sv and B6-ROSA26 components of the polyclonal villus. ( B ) The base of a polyclonal villus with its crypt-villus junction indicated by closed arrows . Three crypts are seen ( open arrows at their base): the one on the left is supplying cells to another villus. The section was incubated with rat anti-β 4 integrin subunit, Cy3 donkey anti–rat Ig, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. Nuclei ( blue ); the β 4 integrin subunit ( orange ); β-gal ( green-brown ). The location of β 4 integrin at the base of epithelial cells and its distribution along the crypt-villus unit are unaffected by ΔN89β-catenin. ( C ) Villi sectioned perpendicular to their crypt-villus axis. The tight junction protein ZO-1 ( orange ) was detected with rat anti-ZO-1 and Cy3 donkey anti–rat Ig. β-Gal ( green ) was visualized with the same reagents used in the preceding sections. The levels and location of ZO-1 in the 129/Sv(ΔN89β-catenin) and B6-ROSA26 components of polyclonal villi are similar (e.g., open arrows ). ( D ) Villi sectioned perpendicular to their crypt-villus axis as in C . The section was incubated with rat anti-β 7 integrin, Cy3-donkey anti–rat Ig, rabbit anti-laminin, rabbit anti–β-gal, FITC donkey anti–rabbit Ig, and bis-benzimide. B6-ROSA26 cells exhibit diffuse staining of their cytoplasm due to the presence of β-gal ( green ). β 7 integrin is confined to intraepithelial lymphocytes ( orange ). Comparable numbers of these cells are seen in the B6-ROSA26 and 129/ Sv(ΔN89β-catenin) components of polyclonal villi and in wholly 129/Sv(ΔN89β-catenin) villi. Laminin appears as linear green immunoreactivity underlying 129/Sv and B6-ROSA26 epithelium (e.g., closed arrows ). The intensity of staining is similar under cells of both genotypes. Bars, 25 μm.

Article Snippet: PLP-fixed frozen sections of jejunum were stained with a 19-member panel of antibodies: ( a ) affinity-purified rabbit anti– Escherichia coli β-galactosidase (β-gal) (1:500; 5′→ 3′ Inc., Boulder, CO); ( b ) rabbit anti– β-catenin sera (see above, final dilution in PBS/blocking buffer = 1:500); ( c ) affinity-purified rabbit anti–c-myc (see above, 1:100); ( d ) affinity-purified rabbit antibodies raised against amino acids 1034–2130 of human APC (APC2, a gift of P. Polakis; ; ); ( e ) affinity-purified rabbit anti–α-catenin (1:500; gift of J. Nelson); ( f ) a monoclonal rat antibody to E-cadherin (1:1,000; Sigma Chemical Co. ; Hermiston et al., 1995 a ); ( g ) rat anti–ZO-1 (polyclonal antibodies, 1:50; Chemicon International, Inc., Temecula, CA); ( h ) rabbit anti-laminin (1: 1,000; Chemicon International Inc.); ( i ) rabbit anti-mouse fibronectin (1: 1,000; Chemicon International Inc.); ( j ) rabbit anti-mouse collagen type IV (1:1,000; Chemicon International Inc.); ( k ) rat anti-mouse β 1 integrin (1:500; PharMingen , San Diego, CA); ( l ) rat anti-mouse β 7 integrin (1:500; PharMingen ); ( m ) rat anti-mouse β 4 integrin (1:500; PharMingen ); ( n ) rat anti-mouse α 6 integrin (1:500; PharMingen ); ( o ) goat anti-BrdU (1:1,000; ); ( p ) rabbit anti-serotonin (1:1,000, a marker of the predominant enteroendocrine subpopulation in the adult mouse intestine; Incstar, Stillwater, MN); ( q ) rabbit anti-chromagranin A (1:1,000, a general marker of enteroendocrine cells; Incstar); and ( r ) rabbit anti-liver fatty acid binding protein (1:1,000, an enterocyte lineage marker; ).

Techniques: Expressing, Cell Differentiation, Transgenic Assay, Staining, Avidin-Biotin Assay, Incubation

Journal: PLoS ONE

Article Title: Low-Dosage Inhibition of Dll4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

doi: 10.1371/journal.pone.0029863

Figure Lengend Snippet: A) Vascular density is increased in granulation tissue of mice treated with sDll4-Fc, being directly proportional to the dosage increase, when compared to controls throughout the experiment. B) Vascular smooth muscle cell coverage is decreased about 3-fold in relation to PBS-injected control mice throughout the experimental days in both tested groups. C,D,E) Representative anti-PECAM and anti-SMA immunofluorescence images of neo-vasculature in granulation tissue of wounds treated with (D) 0,05 mg/kg or (E) 2,5 mg/kg compared with control mice injected with PBS (C), in days 2, 4 and 6. sDll4-Fc therapy leads to decreased smooth muscle cell recruitment and increased vascular density that is directly proportional to the administered dosage. * In graphics represents p<0,05.

Article Snippet: Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA) and rabbit polyclonal anti-mouse α-SMA (Abcam, Cambridge, UK) were used as primary antibodies and species-specific conjugated with Alexa Fluor 488 and 555 (Invitrogen, Carlsbad, CA) were engaged as secondary antibodies.

Techniques: Injection, Immunofluorescence

Journal: PLoS ONE

Article Title: Low-Dosage Inhibition of Dll4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

doi: 10.1371/journal.pone.0029863

Figure Lengend Snippet: Anti-PECAM (red) and anti-SMA (green) immunofluorescence of A) PBS-, B) 0,05 mg/kg sDll4-Fc- or C) 2,5 mg/kg sDll4-Fc-treated mice D,E,F) Representative anti-PECAM immunofluorescence images of neo-vasculature in unaffected skin adjacent to the wound site. G) Vascular density in unaffected skin is not statistically different between PBS-, 0,05 mg/kg- and 2,5 mg/kg-treated mice. The same happens for perivascular cell coverage H). I–J) Hematoxylin and eosin staining of 20 µm cryosections of PBS-, 0,05 mg/kg- and 2,5 mg/kg-treated mice wounds depicting the wound size and unaffected adjacent skin at day 6.

Article Snippet: Rat monoclonal anti-mouse PECAM (BD Pharmingen, San Jose, CA) and rabbit polyclonal anti-mouse α-SMA (Abcam, Cambridge, UK) were used as primary antibodies and species-specific conjugated with Alexa Fluor 488 and 555 (Invitrogen, Carlsbad, CA) were engaged as secondary antibodies.

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

doi: 10.1101/2024.04.01.587641

Figure Lengend Snippet: (A) Schematic view of validating signatures identified from zebrafish myeloid subsets in human burn patient expression profile. (B) Expression level distribution of Immature_Neuts signatures (top panel) and lgals3bpb_Mac1 (bottom panel) by patient TBSA shown as raincloud plots. Paired T-tests were performed between the Low and the Very High TBSA groups. (C) Expression distribution of GPR84 and GYG1 in patients by TBSA level. Spearman correlation (ρ) is calculated between gene expression and TBSA level. (D) Representative flow plots (left) and quantifications of frequencies shown as percentages (right) of GPR84+ neutrophils in burn (n = 6) and healthy (n = 4) blood. Paired samples t-test, p = 0.022.

Article Snippet: Protein-targeted staining was done with the following antibodies in Cell Staining Buffer: Alexa Fluor 700-conjugated anti-human CD16 at 1:400, PE/Fire-640-conjugated anti-human CD66b at 1:400, PerCP-conjugated anti-human CD3 at 1:200, PerCP-conjugated anti-human CD19 at 1:200, PerCP-conjugated anti-human CD56 at 1:400, PerCP-conjugated anti-human CD203c at 1:100 (BioLegend), BUV395-conjugated mouse anti-human CD45 at 1:100 (BD Biosciences), and PE-conjugated GPR84 at 1:200 (Alomone Labs).

Techniques: Expressing, Gene Expression