Journal: Neural Development

Article Title: Axon fasciculation in the developing olfactory nerve

doi: 10.1186/1749-8104-5-20

Figure Lengend Snippet: Primary antibodies

Article Snippet: DBA (Biotinylated) , Vector Laboratories , NA , 1:50.

Techniques: Plasmid Preparation

Journal: RSC Advances

Article Title: Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform †

doi: 10.1039/c8ra05382k

Figure Lengend Snippet: Staining profiles by biotinylated DBA, SBA, HPA and the two forms of MGL in longitudinal sections through fixed murine jejunum. Incubations were done with concentrations of lectins that lead to a strong signal with minimal background (100% level). (a) Negative control by omission of the incubation step with first-step reagent (labelled lectin) with no evidence for lectin-independent signal generation (blank value as shown in ) (b and c) strong reactivity for DBA (b) and SBA (c) was found in the brush border of surface enterocytes from intestinal villi (arrowheads), goblet cells (arrows) and in crypt-associated cells, especially in the deep parts of the crypts (asterisks). (d) Binding of biotinylated HPA was detected supranuclearly in surface enterocytes from intestinal villi (arrowheads) (high-level magnification of this region is shown in an inserted circle above the respective area) and in the deep parts of the crypts (asterisks). (e and f) MGL-dependent positivity (similar for the two protein forms) in crypt-associated cells, most prominently in the crypts' deep parts (asterisks) and comparatively weaker in the cytoplasm of surface enterocytes from intestinal villi. No staining was seen in the brush border (arrowheads; higher magnification is shown in an inserted circle above the respective area). Insets in panels b to e show extent of blocking lectin binding by co-incubation of the lectins with free GalNAc at 200 mM. Concentration of biotinylated lectins used were 1 μg mL −1 for DBA, 3 μg mL −1 for SBA, 1.5 μg for HPA and 4 μg mL −1 for both engineered forms of MGL. Scale bars are 20 μm (a–f) and 5 μm (circles in d and e).

Article Snippet: Biotinylated DBA was obtained from Enzo Life Sciences (Lörrach, Germany), biotinylated HPA from Sigma-Aldrich.

Techniques: Staining, Negative Control, Incubation, Binding Assay, Blocking Assay, Concentration Assay

Journal: RSC Advances

Article Title: Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform †

doi: 10.1039/c8ra05382k

Figure Lengend Snippet: Staining profiles by biotinylated DBA (a–d, arrowheads mark staining of goblet cells in villi) and SBA (e–h, arrows mark staining of crypt-associated cells) in sections of fixed murine jejunum in the presence of increasing concentrations of cognate sugar (GalNAc) assessed in terms of staining intensity (percentage of positive cells). The signal for DBA binding remained at the 100% level in the presence of 1 mM GalNAc presented by compound 3 (a). When raising the sugar concentration stepwisely to 2 mM (b), 5 mM (c) and 10 mM (d), respectively, scaffold-presented GalNAc (by compound 3) led to a notable degree of inhibition. SBA-dependent staining intensity and percentage of positive cells were seen to be reduced more strongly at comparatively low concentrations of 0.001 mM (e), 0.05 mM (f), 0.1 mM (g) and 0.5 mM (h) of GalNAc-presenting compound 2. Semiquantitative grading of staining intensity/percentage of positive cells is given in the rectangular box in the top-right area of each microphotograph (v, surface enterocytes of intestinal villi; c, crypt-associated cells). Intensity of staining in sections is grouped into the following categories: −, no staining; (+), weak but significant staining; ++ medium staining; +++, strong staining; ++++, very strong staining. Percentage of positive cells is expressed in the following eight categories: (1) 0% (no positive cells), (2) < 5% (few positive cells), (3) 5–20%, (4) 21–40%, (5) 41–60%, (6) 61–80%, (7) 81–95% (few negative cells), (8) up to 100% (no negative cells). Concentration of biotinylated DBA/SBA: 1 μg mL −1 /3 μg mL −1 . Scale bars are 20 μm.

Article Snippet: Biotinylated DBA was obtained from Enzo Life Sciences (Lörrach, Germany), biotinylated HPA from Sigma-Aldrich.

Techniques: Staining, Binding Assay, Concentration Assay, Inhibition

Journal: RSC Advances

Article Title: Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform †

doi: 10.1039/c8ra05382k

Figure Lengend Snippet: IC 50 -values of glycoclusters (given in mM based on GalNAc) and the cognate monosaccharide in lectin histochemical assays with biotinylated lectins applied to sections of fixed murine jejunum

Article Snippet: Biotinylated DBA was obtained from Enzo Life Sciences (Lörrach, Germany), biotinylated HPA from Sigma-Aldrich.

Techniques:

Journal: RSC Advances

Article Title: Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform †

doi: 10.1039/c8ra05382k

Figure Lengend Snippet: Staining profiles by the four labelled lectins in longitudinal sections of fixed mouse jejunum in the presence of a constant concentration of cognate sugar (0.02 mM in compound 5; arrows mark staining of the brush border). a–e Signal intensity and percentage of positive cells remained at the 100% level after applying biotinylated DBA (a), whereas both parameters dropped to zero levels when using biotinylated SBA (b). This set-up reduced HPA-dependent binding to nearly 50% level (c). Binding of both forms of MGL was strongly inhibited (d and e). The concentration of 0.2 mM GalNAc presented by the bivalent compound 6 (f–j; arrowheads mark staining of goblet cells) had a slight effect on DBA staining in surface enterocytes (f). This concentration and this type of GalNAc presentation significantly reduced the intensity of the staining patterns of the other lectins applied (for details, please see ). Binding of SBA (g) and HPA (h) was subject to a reduction by about 50% in surface enterocytes of intestinal villi (SBA: brush border, HPA: supranuclearly) and by a less diminished extent of staining intensity in crypt-associated cells. (i and j) The degree of inhibition was strongest for both forms of MGL so that the remaining staining intensity in surface enterocytes of intestinal villi and crypt-associated cells was categorized as weak to medium (for details on categories, please see legend to ). Concentration of biotinylated lectins used were 1 μg mL −1 for DBA, 3 μg mL −1 for SBA, 1.5 μg for HPA and 4 μg mL −1 for the two engineered forms of MGL. Scale bars are 20 μm.

Article Snippet: Biotinylated DBA was obtained from Enzo Life Sciences (Lörrach, Germany), biotinylated HPA from Sigma-Aldrich.

Techniques: Staining, Concentration Assay, Binding Assay, Inhibition

Journal: RSC Advances

Article Title: Revealing biomedically relevant cell and lectin type-dependent structure–activity profiles for glycoclusters by using tissue sections as an assay platform †

doi: 10.1039/c8ra05382k

Figure Lengend Snippet: Three-colour fluorescence staining profiles by DBA, SBA and MGL (CRD + stalk) in longitudinal sections of fixed murine jejunum in the absence or presence of cognate sugar (GalNAc) presented by compound 5 and their (photo)merging. Accessible binding sites for the three lectins were visualised by applying a mixture of biotinylated DBA, Alexa Fluor®-555-labelled SBA (colour assignment to blue) and FITC-labelled MGL (CRD + stalk) (green). Localization of biotinylated DBA became detectable by the second-step reagent Alexa Fluor®-647-conjugated streptavidin (5 μg mL −1 ; colour assignment to red). DAPI was used for staining of nuclei (colour assignment to white). (a–c) Binding of DBA (a) and SBA (b), respectively, was seen in the brush border (arrows) of intestinal enterocytes, in goblet cells (arrowheads) and crypt-associated cells (asterisks). MGL binding was mainly confined to the cytoplasm of surface enterocytes in intestinal villi (c). (d–f) No inhibition of DBA-dependent staining (d) was seen with scaffold (compound 5)-presented GalNAc (0.1 mM), whereas binding of labelled SBA (e) and of MGL (f) was completely inhibited. The three staining patterns were (photo)merged so that binding sites for each lectin in the same section were visualised in the absence (g) and in the presence of cognate sugar (h). The regionally similar DBA- and SBA-dependent signal presence led to overlap (colour change to magenta) in the brush border of intestinal villi, goblet cells and crypt-associated cells (g). No significant overlap, in contrast, was seen with labelled MGL (g). In the presence of compound 5 applied at 0.1 mM GalNAc, DBA-dependent staining was expectably rather insensitive to inhibitor presence, when compared to SBA and MGL (h). Concentration of biotinylated DBA was 1 μg mL −1 , that of Alexa Fluor®-555-labelled SBA was 2 μg mL −1 and FITC-labelled MGL was 16 μg mL −1 . DAPI was used at 0.5 μg mL −1 . Scale bars are 20 μm.

Article Snippet: Biotinylated DBA was obtained from Enzo Life Sciences (Lörrach, Germany), biotinylated HPA from Sigma-Aldrich.

Techniques: Fluorescence, Staining, Binding Assay, Inhibition, Concentration Assay