fluorescein dyes  (Vector Laboratories)

Journal: Human Genetics and Genomics Advances

Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

doi: 10.1016/j.xhgg.2025.100517

Figure Lengend Snippet: The ileal epithelium is dominated by L-PHA + , complex N-glycans in healthy subjects and by truncated, sWGA + N-glycans in active Crohn disease (A) Schematic of N-glycan branching cascade demonstrating the glycan targets of lectins and manganese (Mn) utilization. sWGA (green) stains terminal GlcNAc residues. L-PHA (red) stains complex tri- and tetra-antennary N-glycans. Relative UDP-GlcNAc affinity of glycosyltransferases is also represented (300-fold change from Mgat1 to Mgat5). Figure generated in Biorender, informed by Cummings et al. (ref. ). (B) Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissue paraffin sections, stained for PHA-L (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (PHA-L 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. n = 3–4. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Ratio of sWGA/L-PHA also depicted. Individual data points, mean, and SEM graphed with statistical significance are determined by t test, with p value indicated by two asterisks (<0.01) or three asterisks (<0.001). (C) Volcano plot of glycosyltransferase genes differentially expressed in ileal mucosal biopsies from male and female pediatric patients with severe ileal Crohn disease compared to non-IBD controls. This is a secondary analysis of RNA-seq data (GEO: GSE57945 ). n = 42 controls, n = 63 CD patients. There is an enrichment for MGAT-related pathway members with increased expression of MGAT1 and decreased expression of MGAT3 , MGAT4A/4B , and MGAT5 . MGAT4B is the most highly expressed gene .

Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

Techniques: Glycoproteomics, Generated, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, RNA Sequencing, Expressing

Journal: Human Genetics and Genomics Advances

Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

doi: 10.1016/j.xhgg.2025.100517

Figure Lengend Snippet: Truncated sWGA + N-glycans are increased at the apical/brush border of ileal epithelial cells in ZIP8 391-Thr carriers with Crohn disease Representative lectin immunofluorescence images of ileal biopsies of genotyped patients with active Crohn’s ileitis in (A) ZIP8 391A/391A (non-carriers) and (B) ZIP8 391A/391T (ZIP8 391-Thr heterozygous carriers). Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Scale bar: 50 μm. Asterisk highlights enhanced localization of sWGA staining to the apical membrane/glycocalyx of epithelial cells. To focus on the epithelial compartment, sWGA and L-PHA distribution and overlap were blindly scored by two investigators. Representative images from n = 3 patients/genotype included in figure; histology reviewed and scored for n = 9 ZIP8 391A/391A and n = 6 ZIP8 391A/391T individuals. ns, not statistically significant.

Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

Techniques: Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, Membrane

Journal: Human Genetics and Genomics Advances

Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

doi: 10.1016/j.xhgg.2025.100517

Figure Lengend Snippet: ZIP8 391-Thr-associated defect in N-glycosylation in the ileal epithelial compartment is recapitulated in Zip8 393T-knockin mice (A) Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT), Zip8 +/393T (Het), and Zip8 393T/393T (HM) ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. N = 5 male and female mice/genotype with n = 3–5 fields of view/mice imaged. (B) Quantification of sWGA and L-PHA fluorescence intensity normalized to Hoechst measured using Metamorph. Statistical significance determined by one-way ANOVA, p value indicated by one asterisk (<0.05) or three asterisks (<0.001). (C) Averaged spectra of matrix-associated laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) following on-tissue PNGase F digest to measure the differential abundance of N-glycan species in transverse section of distal ileal tissue of Zip8 +/+ and Zip8 393T/393T mice. Higher m/z species represent tri- and tetra-antennary N-glycan branching. N = 4 male mice/genotype. (D) Confocal laser-scanning triple-label immunofluorescence microscopy images of Jackson C57BL/6 male mice fed purified diet containing variable Mn (<1 ppm = Mn deficient and 2,400 ppm = Mn excess) for 4 weeks. Ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. N = 3 male mice in each group, 2–3 fields of view imaged and quantified per mouse. Scale bar: 50 μm. Individual data points, mean, and SEM graphed, with statistical significance determined by one-way ANOVA and p value indicated by two asterisks (<0.01) or three asterisks (<0.001).

Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

Techniques: Glycoproteomics, Knock-In, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, Mass Spectrometry, Imaging, Purification

Journal: Human Genetics and Genomics Advances

Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

doi: 10.1016/j.xhgg.2025.100517

Figure Lengend Snippet: Oral GlcNAc supplementation restores complex N-glycan branching in intestinal epithelial cells in Zip8 +/393T and Zip8 393T/393T mice Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT) (A), Zip8 +/393T (Het) (B), and Zip8 393T/393T (HM) (C) mice ileal tissue paraffin sections, stained for sWGA (green), L-PHA (red), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. L-PHA and sWGA fluorescence intensity measured using Metamorph. Scale bar: 50 μm. N = 4–5 male and female mice/genotype, with n = 3–7 fields of view/mice imaged. Individual data points, mean, and SEM are graphed. Statistical significance was determined by Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons testing; p value indicated by one asterisk (<0.05), two asterisks (<0.01), three asterisks (<0.001), or four asterisks (<0.0001).

Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

Techniques: Glycoproteomics, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence

Journal: Microsystems & Nanoengineering

Article Title: A temperature-controlled chip holder with integrated electrodes for nanofluidic scattering spectroscopy on highly integrated nanofluidic systems

doi: 10.1038/s41378-025-01125-9

Figure Lengend Snippet: a Schematic of one side of the fluidic system on the 12-inlet chip where one inlet to the sample nanochannel system is filled with 50 mM Fluorescein solution, a second inlet with a 50 mM Brilliant Blue dye solution, and a third inlet with water. The reference fluidic system is filled with water. The empty reservoir will be used as liquid outlet during the later experiments depicted in Figs. & . The inset shows a zoomed-in section at the start of the nanochannel array, where six different sections of a specific sample nanochannel are indicated and color-coded. Each section thus corresponds to a 15 µm long fraction of the in total 120 µm long sample nanochannel. Hence, each section corresponds to 21 binned pixels on the CCD-camera image used for NSS readout from each of the six sections and a sample volume of only 600 attoliter. b Dark field microscopy image of the dashed region marked in ( a ) taken while applying 500 mbar pressure to the Brilliant Blue and Fluorescein reservoirs to enable their mixing at the intersection point of the two microchannels that exit the respective reservoirs. c Pressures at the respective inlets during the dye exchange and mixing experiment. d Time traces of the integrated RSID amplitude measured at 520 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± 5 nm for Fluorescein (see f , j , also Fig. for corresponding RISD and molar extinction coefficient spectra) and at 670 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± 5 nm for Brilliant Blue (see g , Fig. ). This reveals the presence and change in concentration of the respective species in the sample nanochannel. e – j Selected RSID spectra for nanochannel sections 3 and 4 along the experiment timeline that reveal the presence and transient mixing of the dyes at t = 1360 s. The integration intervals used for the time trace in ( d ) are indicated in black. k Ridge plot of the—for clarity inverted—RSID spectra over time revealing the spectral signatures of the two dyes. The transition between them can be seen as a distinct transition from the strong peak at 670 nm (Brilliant Blue) to the lower peak at 520 nm (Fluorescein). We note that while the molar concentrations of the dyes are equal, their molar extinction coefficients are different , which is the reason for the stronger RSID peak of Brilliant Blue

Article Snippet: The Brilliant Blue and Fluorescein dyes were bought as their sodium salts from Merck and diluted into stock solutions of 50 mM concentration with ultrapure water (Milli-Q IQ 7000 water purification, Merck).

Techniques: Microscopy, Concentration Assay

Journal: Microsystems & Nanoengineering

Article Title: A temperature-controlled chip holder with integrated electrodes for nanofluidic scattering spectroscopy on highly integrated nanofluidic systems

doi: 10.1038/s41378-025-01125-9

Figure Lengend Snippet: a To prepare for this experiment, a pressure of 500 mbar was applied to a reservoir filled with 50 mM Fluorescein solution which is connected to one side of the sample nanochannels via a microchannel. At the same time, 800 mbar were applied to a water-filled reservoir on the other side of the sample nanochannel system to establish a water flow through the nanochannels into the Fluorescein-filled microfluidic system. At t = 10 s, all pressures were released, leading to Fluorescein freely diffusing into the water-filled nanochannels, whereof one channel (200 × 200 nm 2 cross-section) was monitored by NSS, divided into six sections indicated and color-coded. b RSID spectra of the six nanochannel sections for 50 °C and at t = 20 s. c – f Time traces of the RSID intensity integrated from 510 to 520 nm (Fluorescein peak) are measured at 22 °C ( b ), 30 °C ( c ), 41 °C ( d ), 50 °C ( e ) for the six color-coded nanochannel sections upon diffusion of the 50 mM Fluorescein solution into water in the channel. Nanochannel section 1 is closest to the Fluorescein- filled microchannel and thus exhibits the strongest response at all temperatures, whereas the RSID-amplitude from channel section 6 closest to the water-filled microchannel stays constant during the whole experiment. Defining the rate of RSID change as a descriptor for the diffusion rate at the four different temperatures, it is evident that steady state is reached faster at the higher temperatures, and that the slope during the transition (indicated with a red dashed line) becomes steeper. g Comparison of the slope values (RSID change per unit time) for the different temperatures, showing that a higher temperature leads to faster diffusion, as expected. The red crosses mark the value of the slope of the red dashed lines fitted to the RSID time traces shown in ( c – f )

Article Snippet: The Brilliant Blue and Fluorescein dyes were bought as their sodium salts from Merck and diluted into stock solutions of 50 mM concentration with ultrapure water (Milli-Q IQ 7000 water purification, Merck).

Techniques: Diffusion-based Assay, Comparison

Journal: Microsystems & Nanoengineering

Article Title: A temperature-controlled chip holder with integrated electrodes for nanofluidic scattering spectroscopy on highly integrated nanofluidic systems

doi: 10.1038/s41378-025-01125-9

Figure Lengend Snippet: We used the same nanofluidic channel with 200 nm × 200 nm cross section and the same experimental scheme at 50 °C as in Fig. , with two electrodes (coated copper wire, 0.5 mm diameter) inserted into the reservoir of the aqueous Fluorescein solution (reservoir 2 in Fig. ) and into the water filled reservoir (reservoir 1 in Fig. ) of the sample nanochannel fluidic system. The cathode (+) is placed in the Fluorescein reservoir while the anode (-) is placed in the water reservoir. Since these two reservoirs are located on either side of the nanofluidic channels on the chip, an electric field can be generated across them, which is oriented such that the (in solution) doubly negatively charged Fluorescein molecules are transported towards the cathode. a RSID amplitude integrated between 523 nm and 533 nm, plotted as a function of time for the six color-coded nanochannel sections for an applied potential of 10 V with the corresponding inverted RSID ridge-plot in ( b ). c , d same as ( a ) but for 5 V applied potential and RSID integration between 527 nm and 537 nm. For 10 V and 5 V the diffusion sets in very slowly after the pressure is released at 10 s, and only reaches a fraction of the RSID at the end of the experiment when compared to Fig. . This is the direct consequence of the electric field counteracting diffusion and thereby preventing Fluorescein molecules from entering the nanochannel. e , f same as ( a ), but for 1 V and RSID integration between 519 nm and 529 nm. Evidently, diffusion of Fluorescein into the water-filled channel sets in directly at 10 s when the applied pressure is released. However, only channel section 1 closest to the Fluorescein-filled microchannel can maintain a relatively high concentration over time. In all other sections, the Fluorescein concentration decreases again because of the applied field that counteracts diffusion. A linear fit has been applied to the RSID slope of section 1 (red dashed line, Fig. ). g , h same as ( a ), but for 0.5 V and RSID integration between 512 nm and 522 nm. Here the observed behavior is very similar to Fig. where no field is applied. Diffusion sets in rapidly at 10 s, when the applied pressures on the system are released, and creates a concentration gradient along the nanochannel. A linear fit has been applied to the RSID slope of section 1 (red dashed line, Fig. ). i Fluorescein RSID spectra measured at 10 V, 5 V, 1 V and 0.5 V applied potential at t = 45 s for the nanochannel section closest to the Fluorescein side (section 1). They reveal a distinct spectral red-shift and intensity decrease of the RSID peak for higher potentials that corresponds to the absorption band of the Fluorescein molecule. This is the consequence of field-induced shifts of the energy levels of the conjugated electron system of Fluorescein (as already demonstrated for other molecules with conjugated electron sytems – ). The position of the peak without voltage applied (as shown in Fig. ) is indicated with a dashed line

Article Snippet: The Brilliant Blue and Fluorescein dyes were bought as their sodium salts from Merck and diluted into stock solutions of 50 mM concentration with ultrapure water (Milli-Q IQ 7000 water purification, Merck).

Techniques: Generated, Diffusion-based Assay, Concentration Assay