fitc labeled mal  (Vector Laboratories)

Journal: Scientific Reports

Article Title: Spatial mapping of influenza and coronavirus receptors in the respiratory and intestinal tract epithelium of beef cattle using advanced PixF image analysis

doi: 10.1038/s41598-025-28429-0

Figure Lengend Snippet: Sialic acid distribution in the beef cattle respiratory tract. ( A – JJ ) Confocal microscopy images of fluorescently labeled lectins Sambucus nigra (SNA, FITC, green) specific for sialic acid (SA) α2,6-Gal, Maackia amurensis II (MAL II, DyLight 650, red) specific for SA α2,3-Galβ (1–3) GlcNAc, and Maackia amurensis I (MAL I, FITC, green) specific for SA α2,3-Galβ (1–4) GlcNAc. Blue represents nuclear staining using DAPI; scale bar = 20 µM. Representative merged images showing SNA/MAL II binding ( A – E ) and MAL I/MAL II binding ( F – J ). Multifocal, apical membranous (white arrow) MAL II labeling was observed on trachea epithelium and extensively on basal cells (orange arrow) ( A ), while goblet cells (arrowhead) showing intense cytoplasmic SNA ( Ai ) and MAL II ( Aii ) labeling. Image analysis of tracheal epithelial lining (3D plot of insert in A ) showing greater MAL II labeling even in co-localized areas (arrow), and slightly higher SNA in goblet cell (arrowhead) ( Aiii ). Sub-mucosal areas showing both SNA and MAL II ( B ), with higher SNA in lamina propria connective tissue (asterisk) and higher MAL II in glands (dashed white outline). The 3D plot of insert in ( B ) demonstrated varying levels of expression of MAL II (arrow) and SNA (arrowhead) ( Biii ). Goblet cells and epithelial lining of the bronchus, bronchiole, and alveoli show intense MAL II labeling (arrow) ( C , D , E ) with SNA labeling in the lamina propria of the bronchus (white asterisk) ( C ) and the interstitial region of the alveolar wall (asterisk) ( E ). Quantitative image analysis confirmed that the lining of epithelial cells has higher membranous MAL II labeling (arrow) ( Ciii , Diii , Eiii ). MAL I demonstrated a labeling distribution analogous to that of MAL II, albeit with attenuated signal intensity ( F , H – J ), reflected in quantitative image analysis ( Fiii , Hiii – Jiii ). MAL I is more robust in the submucosal glands of the trachea (dashed white outline) ( G , Giii ). ( K – O ) Confocal images of the respiratory tract showing SA N-glycolylneuraminic acid (Neu5Gc, FITC, green) following immunofluorescence staining. Neu5Gc was detected on the apical membrane and cell borders of ciliated pseudostratified epithelia of the trachea ( K ), multifocally on the epithelial lining of the bronchus ( M ), bronchiole ( N ), and the alveoli ( O ). The sub-epithelial glands in the tracheal region also showed Neu5Gc ( L ).

Article Snippet: The sections were then incubated overnight (16 h) at 4 °C in a humidified chamber with optimized concentrations of 10 μg/ml fluorescein isothiocyanate (FITC) labeled SNA (Cat# FL-1301-2, Vector Laboratories) or 10 μg/ml FITC labeled MAL I (Cat# FL-1311-2, Vector Laboratories), and 5 μg/ml MAL II (Cat# B-1265-1, Vector Laboratories).

Techniques: Confocal Microscopy, Labeling, Staining, Binding Assay, Expressing, Immunofluorescence, Membrane

Journal: Scientific Reports

Article Title: Spatial mapping of influenza and coronavirus receptors in the respiratory and intestinal tract epithelium of beef cattle using advanced PixF image analysis

doi: 10.1038/s41598-025-28429-0

Figure Lengend Snippet: Sialic acid distribution in the beef cattle intestinal tract. (A-H) Confocal microscopy images of fluorescently labeled lectins Sambucus nigra (SNA, FITC, green) specific for sialic acid (SA) α2,6-Gal, Maackia amurensis II (MAL II, DyLight 650, red) specific for SA α2,3-Galβ (1–3) GlcNAc, and Maackia amurensis I (MAL I, FITC, green) specific for SA α2,3-Galβ (1–4) GlcNAc. Blue represents nuclear staining using DAPI; scale bar = 20 µM. Representative merged images showing SNA/MAL II binding ( A – D ) and MAL I/MAL II binding ( E – H ). The epithelial lining of the small and large intestine shows MAL II labeling (insets of A , C ), which was evident in the quantitative image analysis (arrow) ( Aiii , Ciii ). Goblet cells located in the crypt and apical region of the intestines show abundant MAL II labeling ( A – D ). SNA labeling was limited to lamina propria (asterisk) ( A , B ) and goblet cells in the crypt region of the intestines (arrowhead) ( B , D ). MAL I and MAL II were labeled largely co-localized ( E – H ) in both the epithelial lining and goblet cells. Quantitative image analysis indicates MAL II was comparatively higher in the co-localized areas (arrow) (insets of E , G , Eiii , Giii ). Uniform labeling of MAL I in goblet cells distributed toward apical or crypt regions of the small intestine ( Ei , Fi ). Note the gradient decrease in MAL I labeling in the goblet cells distributed toward the apical to crypt regions of the large intestine ( Gi , Hi ). ( I – L ) Confocal images of small and large intestines showing SA N-glycolylneuraminic acid (Neu5Gc, FITC, green) following immunofluorescence staining. The SA Neu5Gc was expressed on the epithelial lining and goblet cells of the small intestine ( I , J ). Neu5Gc expression was more scattered in the large intestine, and there was no evident expression on the epithelial lining ( K , L ).

Article Snippet: The sections were then incubated overnight (16 h) at 4 °C in a humidified chamber with optimized concentrations of 10 μg/ml fluorescein isothiocyanate (FITC) labeled SNA (Cat# FL-1301-2, Vector Laboratories) or 10 μg/ml FITC labeled MAL I (Cat# FL-1311-2, Vector Laboratories), and 5 μg/ml MAL II (Cat# B-1265-1, Vector Laboratories).

Techniques: Confocal Microscopy, Labeling, Staining, Binding Assay, Immunofluorescence, Expressing

Journal: eBioMedicine

Article Title: Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone

doi: 10.1016/j.ebiom.2024.105163

Figure Lengend Snippet: Sialic acid blockade can prevent/inhibit prostate cancer bone metastasis. ( a ) Inhibition of sialylation in TRAMPC2 cells using P-SiaFNEtoc detected using pan-specific Lectenz lectin flow cytometry. Cells were treated with a range of concentrations of P-SiaFNEtoc inhibitor from 2 μM to 512 μM for 72 h. The intensities were normalised to a DMSO control. ( b ) Detection of α2-6 linked sialylated N -glycans in TRAMPC2 cells using SNA lectin flow cytometry. TRAMPC2 cells treated with 64 μM P-SiaFNEtoc for 72 h had reduced levels of SNA binding indicating a reduction in α2-6 linked sialylation in these cells (unpaired t test, p = 0.0001). ( c ) Luciferase tagged TRAMPC2 cells (control or pre-treated with 64 μM P-SiaFNEtoc for 72 h) were injected into immunocompetent C57BL/6 mice via sub-cutaneous injection and tumours were monitored using in vivo bioluminescence imaging. Pre-treatment of TRAMPC2 cells with P-SiaFNEtoc (which removed sialylated glycans) significantly reduced tumour burden over 6 weeks (n = 10, Mann–Whitney test, p = 0.0233) thus suggesting that sialic acid blockade has the potential to inhibit the growth of prostate tumours. ( d ) Inhibition of sialylation in RM1 cells using P-SiaFNEtoc detected using pan-specific Lectenz lectin flow cytometry. Cells were treated with a range of concentrations of P-SiaFNEtoc inhibitor from 2 μM to 512 μM for 72 h. The intensities were normalised to a DMSO control. ( e ) Detection of α2-6 linked sialylated N -glycans in RM1 cells using SNA lectin flow cytometry. RM1 cells treated with 256 μM P-SiaFNEtoc for 72 h had reduced levels of SNA binding indicating a reduction in α2-6 linked sialylation in these cells (unpaired t test, p < 0.0001). ( f ) Luciferase tagged RM1 cells (control or pre-treated with 256 μM P-SiaFNEtoc for 72 h) were injected into immunocompetent C57BL/6 mice via intra cardiac injection. Tumours were monitored over 15 days using in vivo bioluminescence imaging. ( g , h ) Pre-treatment of RM1 cells with P-SiaFNEtoc (to remove sialylated glycans) significantly reduced the number of skeletal tumours formed (Mann–Whitney test, p = 0.0454), the incidence of tumour in left tibias (Chi-square test, p = 0.0455), and significantly increased survival time in mice (Log-rank test, p = 0.012). ( i ) Micro-CT analysis demonstrated that P-SiaFNEtoc significantly alleviated bone destruction in the trabecular bone of tibias and increased trabecular bone volume (BV/TV, p = 0.0211) and trabecular number (Tb. N, p = 0.035) (n = 9, unpaired t test, ∗p < 0.05). Representative images are shown. Scale bar is 200 μm.

Article Snippet: Slides were incubated for 3 h at room temperature with FITC-conjugated SNA lectin (Vector labs, FL-1301-2) at 1:500 or FITC-conjugated MAL I Lectin (Vector labs, FL-1311-2) at 1:500.

Techniques: Inhibition, Flow Cytometry, Control, Binding Assay, Luciferase, Injection, In Vivo, Imaging, MANN-WHITNEY, Micro-CT