fibronectin rabbit pab  (Proteintech)

Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A-B) 2D-projected confocal images of the near-blastopore region (orange arrowhead) at stages 18 and 24. Schematics indicates approximate imaging locations and plane (Blue shaded box). (A) En face views of F-actin, keratin 8, and fibronectin and fibronectin in parasagittal section. The sagittal section is rotated 90° to align the blastopore with en face images. The regions of tissue rotation are located approximately 100 to 150 µm from the center of the blastopore. (B) En face views of keratin 8 and fibronectin, with a zoomed inset (yellow box). Aligned fibronectin fibrils are most prominent within 80 to 120 µm of the blastopore. (C) Polar histograms (rose plots) of fiber orientations at stage 18 and 24 (n, number of embryos used; Square goodness-of-fit test, ∗∗∗∗p<0.0001) (D) Workflow for image processing and analysis of immunostained samples. (E) Fibronectin morphological features from stage 18 and stage 24. (C and E) measured within a 100 by 100 µm region ventral to the blastopore. Each symbol represents the mean value per embryo (Mann-Whitney U, ∗p=0.02). Bars; mean ± 95% CI. (A-B) scale bars, 20 µm. (A-B) Xenopus illustrations © Natalya Zahn (2022).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A) Dorso-anterior index (DAI) of the severity of dorsalized and ventralized phenotypes. (B) Representative phenotypes of each treatment at stage 20, showing characteristic morphological differences between dorsalized, ventralized, and control embryos. (C) Final frame of brightfield timelapse sequence, overlaid with a yellow deformation map (see also Video S6). (D) Vorticity overlaid randomized dot plots deformed by calculated displacements, and max-projected across all timepoints to visualize movement patterns. Representative images are shown for each treatment. (E) SWIRL predicted vortices highlight vortex structure and organization. (F-G) Quantitative comparison of predicted vortex characteristics in dorsalized (8 vortices from 11 embryos), control (32 vortices from 19 embryos), and ventralized (11 vortices from 11 embryos) embryos. Each symbol represents a single predicted vortex (Mann-Whitney U, ∗∗p=0.0012; ∗∗p=0.0061; ∗∗∗p=0.0002; ∗∗∗∗p<0.0001). (F) Vortex compactness (mean ± 95% CI). (G) Vortex swirling strength (mean ± 95% CI). (H) Fibronectin networks in posterior tissues from stage 24 embryos within a 100 by 100 µm region ventral to the blastopore. (I) Fibronectin orientation frequency in each treatment (n, number of embryos used; Square goodness-of-fit test, ∗∗∗∗p<0.0001) (J) Morphological features of fibronectin network in dorsalized, control, and ventralized embryos. Each symbol represents the mean value per embryo (Mann-Whitney U, ∗p=0.0426; ∗∗∗∗ p < 0.0001). Bars indicate mean ± 95% CI. (B-C) scale bars, 100 µm; 20 µm in (H). (A) DAI diagram with permission of the publisher. Xenopus illustrations © Natalya Zahn (2022).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Control, Sequencing, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A) Methods used for targeted disruption of fibronectin organization and integrin-fibronectin interactions. (B) Representative phenotypes showing morphological changes following fibronectin disruption. (C) 2D max-projected confocal image of fibronectin within a 100 by 100 µm region ventral to the blastopore. (D) Morphological features of fibronectin matrix for control (mAb 4H2) and function-blocking (mAb P8D4) treatments. Each symbol represents the per-embryo mean (Mann-Whitney U, ∗p=0.0350; ∗∗p=0.0023). Bars indicate mean ± 95% CI. (E) Final frame of brightfield timelapse sequence overlaid with yellow deformation map (see also Video S7). (F) Time-projected displacement of random dot plot overlaid with vorticity. Disruptions to fibronectin result in less distinct or absent bi-directional vortices compared to controls. (G) SWIRL predicted vortex structure and spatial distribution across treatments. (H-J) Vortex characteristics across treatments. Each symbol in (H) and (I) represents a single predicted vortex: 4H2 (19 vortices from 10 embryos), P8D4 (8 from 11), COMO (21 from 13), and FNMO (18 from 13). In (J), each symbol represents an embryo with a predicted vortex pair. Vortex formation was significantly disrupted in P8D4- and FNMO-treated embryos, with a reduction in detected vortices and alterations in vortex compactness (H) and swirling strength (I). (J) Vortex asymmetry index (Mann-Whitney U, ∗p<0.0290; ∗∗p=0.0054; ∗∗p=0.0024; ∗∗∗p=0.0002). Bars indicate mean ± 95% CI, except in (H). Scale bars, 100 µm in (B,E); 20 µm in (C).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Disruption, Control, Blocking Assay, MANN-WHITNEY, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Non-typeable Haemophilus influenzae major outer membrane protein P5 contributes to bacterial membrane stability, and affects the membrane protein composition crucial for interactions with the human host

doi: 10.3389/fcimb.2023.1085908

Figure Lengend Snippet: Analysis of bacterial pathogenic phenotypes and membrane stability. (A) Bacterial adherence to human type II alveolar epithelial cells (A549) at multiplicity of infection (MOI) of 100 for 30 min. Mean data from three independent experiments (biological replicates) is presented. Bacterial adherence was presented as percentage of CFU recovered per well relative to initial inoculum. (B) Binding of NTHi 3655 wild-type and mutants to human fibronectin. Bacterial (5×10 7 CFU) binding to human fibronectin (0.8-2.0 µg/ml) in 100 µl reactions was analysed by flow cytometry after incubation for 1 hour at 37°C. Rabbit anti-human fibronectin and FITC-conjugated swine anti-rabbit pAbs were used to detect the bacterial-bound fibronectin. Data represent mean values of three independent experiments. (C) Serum killing of NTHi 3655 wild-type and mutants. Bacterial (1.5×10 3 CFU) killing by 5% NHS was analysed by CFU count on chocolate agar. Heat-inactivated serum was included as a negative control and here no bacteria were killed (data not shown). Percentage of bacterial survival was expressed as (T t CFU/T 0 CFU)×100. T 0 represents CFU of sample plated at 0 min; and Tt represents CFU of sample plated at indicated time points. Data represent mean values of three independent experiments. (D) Outer membrane vesicles (OMVs) production among NTHi 3655 wild-type and mutants. OMVs from bacterial cultures were sucrose-density gradient purified and subjected to nanoparticle tracking analysis with a NanoSight NS300. OMV samples were diluted in PBS until 20-120 particles per frame were archived. Settings were optimized using 100nm polystyrene beads, and samples were recorded using the same settings (camera level 12, three recordings of 30 sec each). Recordings were thereafter processed using the NanoSight 3.1 software. Data represents mean values from three independent experiments. (E) Spot viability assay of bacterial survival in response to hyperosmotic environment. Bacteria that were serially diluted (10 9 to 10 4 CFU/ml) was spotted on chocolate agar without sodium chloride (NaCl) (left panel) or supplemented with 50 mM (middle panel) and 100 mM NaCl (right panel). Images were captured using ProtoCOL 3 HD (Synbiosis, UK). The assay was repeated in three independent experiments, and images from a representative experiment were shown. For panel A-D, error bars indicate standard deviations. Differences between wild-type and mutants were calculated by one-way ANOVA for panel (A, D) ; and two-way ANOVA for panel (B, C) *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.001. WT, NTHi 3655 wild-type; Δ p5 , p5 -knockout mutant (NTHi 3655Δ p5 ); Δ p5 CTD , mutant expressing P5 without CTD (NTHi 3655Δ p5 CTD ); Δ p5::p5, p5- transcomplemented NTHi (NTHi 3655Δ p5::p5 ).

Article Snippet: Bacteria-bound fibronectin was detected with rabbit anti-human fibronectin pAb (Dako, Glostrup, Denmark) and FITC-conjugated swine anti-rabbit pAb (Dako).

Techniques: Infection, Binding Assay, Flow Cytometry, Incubation, Negative Control, Purification, Software, Viability Assay, Knock-Out, Mutagenesis, Expressing

Journal: Frontiers in Immunology

Article Title: IL-22 regulates endometrial regeneration by enhancing tight junctions and orchestrating extracellular matrix

doi: 10.3389/fimmu.2022.955576

Figure Lengend Snippet: Antibodies.

Article Snippet: 9 , Fibronectin, rabbit polyclonal , Bioss, MA, USA , bs-0666R , 1:1,000.

Techniques: Concentration Assay