lectin dye rhodamine ricinus communis agglutinin i  (Vector Laboratories)

Journal: bioRxiv

Article Title: 2-D organoids demonstrate specificity in the interactions of parasitic nematodes and their secreted products at the basal or apical intestinal epithelium

doi: 10.1101/2024.12.16.628471

Figure Lengend Snippet: A) Schematic of experimental culture conditions for the generation of 2-D enteroid cultures in Matrigel-coated transwell plates. B) Representative fluorescence microscopy image showing complete transwell coverage by 2-D enteroid monolayer stained with 4’,6-diamindino-2-phenylindole (DAPI) for nuclei (blue). C) Representative confocal immunofluorescence image showing organisation of Ki67+ Alexa Fluor 647 (AF647, yellow) cells into proliferative loci, surrounded by terminally differentiated goblet cells labelled with UEA & SNA FITC (green). Nuclei are stained with DAPI (cyan) and F-actin labelled with phalloidin (Magenta). D) Z-stack projection of confocal immunofluorescence image demonstrating apical brush border of epithelial cells by co-staining of microvilli marker Villin AF647 (Magenta) and F-actin using Phallodin AF594 (White). Ulex europaeus agglutinin I (UEA) fluorescein (FITC) and Sambucus nigra Lectin (SNA) FITC binds lectins present in mucus of goblet cells, UEA & SNA FITC (green) can be seen emerging through the brush border. E-I) Representative fluorescence microscopy images of 2-D enteroids with DAPI-stained nuclei, phalloidin AF594 binding F-actin, and UEA/SNA labelling goblet cells, and antibody-based immunohistochemistry in AF647 of E) villin on microvilli of enterocytes, F) the Ki67+ stem and transit-amplifying cells, G) lysozyme 1-expressing Paneth cells, H) Dckl1 expression on tuft cells and I) chromogranin A on enteroendocrine cells.

Article Snippet: Permaebilisation/block buffer was removed and samples were incubated with primary antibodies α-Lysozyme 1 (1:40, DAKO, A0099), α-DCAMKL1 (Abcam, UK, ab31704), α-Villin (1:100, Abcam, ab130751), α-Chromogranin-A (1:50, Abcam, ab15160), α-Ki67 (1:250, Abcam, ab16667) and the lectin dyes Sambucus Nigra FITC (SNA 1:50, Vector laboratories, UK, FL-1301) and Ulex europaeus FITC (UEA 1:1000, Sigma-aldrich, 19337) in diluent (PBS, 5% BSA, 0.25% Triton-X) overnight at 4°C.

Techniques: Fluorescence, Microscopy, Staining, Immunofluorescence, Marker, Binding Assay, Immunohistochemistry, Expressing