griffonia simplicifolia i  (Vector Laboratories)

Journal: bioRxiv

Article Title: Maternal high-fat, high-sucrose diet-induced excess adiposity is linked to placental hypoxia and disruption of fetoplacental immune homeostasis in late gestation

doi: 10.1101/2024.09.30.615691

Figure Lengend Snippet: (a) Representative composite images of H&E-stained placentas. Boundaries between Labyrinth (Lab), Junctional Zone (JZ), and maternal tissue (decidual basalis (DB) and myometrium (Myo)) were defined based on tissue architecture (hashed lines); scale bar = 1000 µm. (b) Cross sectional areas of the total placenta (combined JZ and Lab), individual compartments, (c) area ratios, and proportional areas were measured for placentas from CON and HFHS pregnancies. (d) Representative multi-channel overlay of fields from the Labyrinth of CON and HFHS placentas co-stained for the vascular basement membrane and perivascular cells (pericytes) using isolectin beta 4 from Griffonia Simplicifolia (GS-Iβ4, magenta), anti-alpha smooth muscle actin monoclonal antibody (α-SMA, green), respectively; scale bar = 100 µm. (e) Proportional area within the Labyrinth occupied by fetoplacental vasculature, defined using combined staining of GS-Iβ4 and α-SMA (Fetal Vascular Area). (f) Stability of fetoplacental vasculature measured according to the percent of the Fetal Vascular Area occupied by α-SMA positive staining Data are presented with boxplots depicting the min and max values within 1.5 IQR (whiskers), IQR (box), and median (center line). Large data points represent the mean of replicate measures from sample replicates, small data points depict individual values of each sample replicate. Data were analysed using two-way ANOVA on mixed effects linear models with diet and sex as fixed effects and sample (placenta) as a random effect. P-values are indicated where main effects were statistically significant. Boxplots without shared letters are significantly different from one another according to post-hoc comparison of estimated marginal means. All samples were analysed in triplicate for CON (n = 5 – 6/sex) and HFHS (n = 8/sex) placentas. All samples are taken from composite images imaged at 20× magnification.

Article Snippet: Placentas were fluorescently co-stained for labyrinth pericytes using a monoclonal anti-alpha smooth muscle actin antibody (α-SMA; clone 1A4, Abcam), and the vascular basement membrane using Isolectin β4 from Griffonia Simplicifolia (GS-Iβ4, Vector Laboratories) to identify fetal vascular structures within the placenta ( , ).

Techniques: Staining, Membrane, Comparison

Journal: bioRxiv

Article Title: Maternal high-fat, high-sucrose diet-induced excess adiposity is linked to placental hypoxia and disruption of fetoplacental immune homeostasis in late gestation

doi: 10.1101/2024.09.30.615691

Figure Lengend Snippet: (a) H&E staining of CON and HFHS placental tissues depicting representative histopathology of CON and HFHS placentas. The presence of tissue lesions consistent with occlusions/calcification (black arrows) and eosinophilic fibrinoid deposition (white arrows) were more frequently observed in HFHS samples; scale bar = 100 µm. (b) Representative composite images of tissue sections from CON (n = 5 – 6/sex) and HFHS (n = 8/sex) stained for calcium deposition using Alizarin Red S. Hashed outlines indicate the labyrinth zone, to which analysis was restricted. White arrows indicate calcified foci; scale bar = 1000 µm. (c) Percent of placental samples with the presence of calcium in at least one replicate tissue section (3 replicates/sample). (d) Quantification of calcification severity based on the percent Alizarin Red S positive area; images depict representative samples each quartile of Alizarin Red S staining within the HFHS group (both males and females); scale bar = 1000 µm. (e) Representative fields of the labyrinth from serial placental sections stained for calcium (left panel), placental vascular endothelium (GS-Iβ4, CD31; middle panel), or syncytiotrophoblast and macrophages (SLC16A1 and F4/80, respectively; right panel); scale bar = 100 µm. (f) Correlation of mean placental labyrinth calcification and fetoplacental vascular proportional areas in HFHS female (magenta) and male (teal) samples. Data in (c) are presented with boxplots depicting the IQR (box), median (center line) and min and max values within 1.5 IQR for mean values (whiskers); large data points represent the mean value of triplicate measures per sample, small data points represent individual replicates. Data points in (f) represent the mean value for each sample. All representative image fields were taken from composite stitched images of whole tissue sections imaged at 20× magnification. Data were analysed using Fisher’s exact test with post-hoc Bonferroni correction for multiple testing (b), two-way ANOVA on mixed effects linear models (c) or Spearman correlation (f).

Article Snippet: Placentas were fluorescently co-stained for labyrinth pericytes using a monoclonal anti-alpha smooth muscle actin antibody (α-SMA; clone 1A4, Abcam), and the vascular basement membrane using Isolectin β4 from Griffonia Simplicifolia (GS-Iβ4, Vector Laboratories) to identify fetal vascular structures within the placenta ( , ).

Techniques: Staining, Histopathology

Journal: bioRxiv

Article Title: Molecular differences between neonatal and adult stria vascularis from organotypic explants and transcriptomics

doi: 10.1101/2024.04.24.590986

Figure Lengend Snippet: P30 cross section of a cochlear turn (A) and P0 wholemount stria vascularis (B) stained with Phalloidin for actin (green) Lectin for blood vessels (magenta) and DAPI for nuclei (blue). Orthogonal view of the wholemount SV in bottom panel of (B) marks the nuclei of the three cell layers of the SV (white dashed lines; m= marginal cells, i = intermediate cells, b = basal cells). Scale bars in A: 50 µm in low magnification images, 10 µm in high magnification images. Scale bars in B: 50 µm. C) Side-by-side differential interference contrast imaging of neonatal and adult SV organotypic explants. SV were dissected and cultured on Matrigel-coated plates in standard conditions for 72 h. Scale bar: 1 mm.

Article Snippet: Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I), Fluorescein (1:250; Vector Laboratories FL-1101-2), or Rhodamine (Vector Laboratories RL-1102-2) was used to stain blood vessels.

Techniques: Staining, Imaging, Cell Culture

Journal: bioRxiv

Article Title: Molecular differences between neonatal and adult stria vascularis from organotypic explants and transcriptomics

doi: 10.1101/2024.04.24.590986

Figure Lengend Snippet: (A-C) Representative images of organotypic stria vascularis explants at P0, P7 and P30 cultured with the proliferation marker, BrdU (red), over 72 h. (A’-C’) Merged images of BrdU + nuclei and DAPI stained nuclei (blue). Scale bars: 1 mm in A-B’; 200 µm in C-C’. (D) Quantification of BrdU + nuclei. A one-way ANOVA was performed, F(2, 25) = 34.15, p < 0.0001. Tukey’s multiple comparisons test was performed for post-hoc analysis. (E-F”) Cryosections of the basal turn of the P0 and P30 cochlea were stained with Ki67 (green), Lectin (magenta) and DAPI. Scale bars: 100 µm for low magnification images; 50 µm in high magnification images. (G) Quantification of Ki67 in P0 and P30 SV using RT-qPCR. A two-tailed unpaired t-test was performed, t(4) = 70.16, p < 0.0001. *p = .0235, ****p < .0001. All graphs show mean with SEM.

Article Snippet: Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I), Fluorescein (1:250; Vector Laboratories FL-1101-2), or Rhodamine (Vector Laboratories RL-1102-2) was used to stain blood vessels.

Techniques: Cell Culture, Marker, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Oncogenesis

Article Title: Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway

doi: 10.1038/s41389-023-00494-y

Figure Lengend Snippet: A GSL-II lectin pull-down assay for integrin β1. Proteins in cell lysates from B4GALT1 knockdown PLC5 and HA22T cells and B4GALT1 knockout PLC5 cells were pulled down using GSL-II agarose beads and immunoblotted using an anti-integrin β1 (ITGB1). Three independent siRNAs for B4GALT1and two independent B4GALT1 knockout clones were used, as indicated. B GSL-II lectin pull-down assay for integrin α6. Proteins in the cell lysates from B4GALT1 knockdown PLC5 and HA22T cells and B4GALT1 knockout PLC5 cells were pulled down using GSL-II agarose beads and immunoblotted using an anti-integrin α6 (ITGA6). C B4GALT1 predominantly modified N-glycans on integrin β1. PLC5 cell lysates (0.5 mg) were treated with PNGase F and then pulled down (PD) using GSL-II lectin. The pull-down proteins were separated using 6% SDS-PAGE and analyzed via immunoblotting with anti-ITGB1. D B4GALT1 predominantly modified N-glycans on integrin α6. PLC5 cell lysates (0.5 mg) were treated with PNGase F and then pulled down (PD) using GSL-II lectin. The pulled-down proteins were separated using 6% SDS-PAGE and analyzed via immunoblotting using anti-ITGA6 (A) or anti-ITGA6 (B) antibody. Representative results from three independent experiments are shown. Data are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t -test.

Article Snippet: For the lectin pull-down assay, cell lysates (1 mg) were incubated with Griffonia Simplicifolia Lectin II (GSL-II) biotinylated (Vector Laboratories) at 4 °C for 16 h. The next day, the sample was incubated with streptavidin beads (Vector Laboratories) for 2 h at room temperature.

Techniques: Pull Down Assay, Knockdown, Knock-Out, Clone Assay, Modification, SDS Page, Western Blot