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: STAR Protocols

Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment

doi: 10.1016/j.xpro.2023.102283

Figure Lengend Snippet:

Article Snippet: Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) , Vector Labs , RL-1112.

Techniques: Labeling, Plasmid Preparation, Quadruple Knockout, Recombinant, Microscopy, Software, Sterility, Membrane