Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: Examples of engraftment of haematopoietic lineage marrow cells (HLMCs) and mesenchymal stem cells at 3 days after HgCl2. (a) GFP‐positive control shows proximal tubular cells (PHA‐L, blue colour) co‐stained for GFP (brown colour). (b) GFP‐negative control (wild type) showing no detection of GFP. (c) BMT mouse treated with HgCl2 demonstrating GFP‐positive proximal tubular epithelial cells. Dashed black arrows indicate PHA‐L stained donor HLMC‐derived tubular cells. (d) Male positive control; Y chromosomes were detected by indirect FISH, black arrows point to Y chromosomes (brown dot) in proximal tubular cells histochemically stained with PHA‐L (red colour). (e) Female control showing the lack of Y chromosome detection within proximal tubular cells histochemically stained with PHA‐L. (f) Scattered Y chromosomes (brown dot) in renal interstitial area, not in proximal tubular cells. (g) High magnification of yellow box in (f); a–f, ×400; g, ×600. BMT, bone marrow transplantation; FISH, fluorescence in situ hybridization; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Positive Control, Staining, Negative Control, Derivative Assay, Transplantation Assay, Fluorescence, In Situ Hybridization

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: (a) Changes in the abundance of GFP‐positive cells within the PHA‐L stained cell population in control mice and mice treated with HgCl2 (n = 5 per treatment time point). *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. The percentages of GFP‐positive cells were adjusted based on a correction factor derived from the actual cell count of 41% of PHA‐L stained cells being GFP‐positive in GFP donor mice. (b) Changes in the 3H‐LI of PHA‐L stained proximal tubular cells of: combined indigenous and donor HLMC (left panel); indigenous origin (central panel); donor HLMC origin (right panel). n = 5 per group. *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. (c–g) Examples of chimerism and proliferation (3H‐thymidine labelling) of GFP+ HLMC‐derived PHA‐L‐stained cells at 3 days after HgCl2. (c) Black arrowheads indicate PHA‐L stained (blue colour in tubular cell apical membrane) donor GFP+ HLMC‐derived tubular cells (brown colour in cytoplasm) under bright field, and black arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells, ×500. (d) The same field under dark field, white arrowheads point to silver grains (3H‐thymidine labelling) and white arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells (×500). (e) Bright field and (f) dark field are the higher magnification of yellow box area in (c). (g) The images in (e) and (f) were combined to help to show silver grains over cells that are donor GFP+ HLMC‐derived PHA‐L‐stained tubular cells. GFP, green fluorescent protein; HLMC, haematopoietic lineage marrow cell; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Staining, Derivative Assay, Cell Counting

Journal: Journal of molecular endocrinology

Article Title: Stomach gastrin is regulated by sodium via PPAR-α and dopamine D 1 receptor

doi: 10.1530/JME-19-0053

Figure Lengend Snippet: Human stomach antrum and G cell immunochemistry for gastrin and PHA-L. (A) Gastrin, phytohemagglutinin-leucoagglutinin (PHA-L, marker of complex type carbohydrate residues which are expressed in gastric parietal cells), and nuclei (blue) in the antrum of fresh human stomach. Scale bar = 10 μm. (B) Gastrin and PHA-L staining in G cells isolated from human stomach and gastrin staining in SW626 cells (ovarian adenocarcinoma cells). G cell- and SW626 cell-control using non-specific rabbit IgG antibody show almost no staining. NG = negative control, non-specific rabbit IgG antibody. Scale bar = 10 μm.

Article Snippet: The cells were then stained with anti-goat gastrin antibody (Santa Cruz) or anti-rabbit antibodies to various targets (gastrin, DAKO 1:200), complex type carbohydrate residues marker Phaseolus vulgaris -leucoagglutinin (PHA-L, Vector Laboratories), D 1 R (generated in our laboratory) ( Sanada et al. 1999 ), D 5 R (Santa Cruz, 1:100), tyrosine hydroxylase (TH, Cell Signaling, 1:200), and DOPA decarboxylase (DDC, Millipore, 1:200) for 2 h at room temperature.

Techniques: Marker, Staining, Isolation, Negative Control

Journal: BioTechniques

Article Title: Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay.

doi: 10.2144/000114208

Figure Lengend Snippet: Figure 4. Lectin binding pattern of urinary EVs from healthy subjects. Immobilized uEVs were allowed to react with biotinylated plant lectins for 30 min at RT. The unbound material was washed out, followed by addition of avidin/biotinylated HRPO and TMB solution. Optical density was monitored at 450 nm. Binding of each lectin was normalized to binding of anti-CD63 antibody, which was run as the control for uEV immobilization. The results are mean values of three experiments. uEVs from both male and female donors were analyzed. Error bars represent the standard deviation. Lectins: Con A (specific for high mannose-type, hybrid-type, and biantennary complex type N-glycans), WGA (specific for GlcNAcb1.4 oligomers/Galb1,4GlcNAc), RCA (specific for Galb1,4GlcNAc), SNA (specific for sialic acid a2,6-Gal), MAA (specific for sialic acid a2,3-Gal), PHA-E (specific for bisected biantennary chains), PHA-L (specific for tri/tetraantennary complex-type N-glycans), LCA (specific for bi/triantennary complex-type with core fucose), PNA (specific for Galb1,3GalNAca-Ser/Thr, T antigen), VVL (specific for terminal GalNAca-Ser/Thr, Tn antigen/ GalNAc1,3Gal), DBL (specific for a1,3GalNAc and blood group A antigen).

Article Snippet: Biotinylated plant lectins [concanavalin A (Con A), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Maackia amurensis lectin II (MAL II), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA I), Vicia villosa lectin (VVL), and Dolichos biflorus agglutinin (DBA)], VECTASTAIN ABC Kit, biotinylated goat anti-mouse IgG, and biotinylated rabbit anti-goat IgG were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Binding Assay, Avidin-Biotin Assay, Control, Standard Deviation

Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L9 , Biotinylated Phaseolus vulgaris Erythroagglutinin , PHA-E , Vector Laboratories , BK-2000.

Techniques: Plasmid Preparation, Positive Control

Journal: PLoS ONE

Article Title: Chemical Characterization of N -Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4

doi: 10.1371/journal.pone.0133784

Figure Lengend Snippet: The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.

Article Snippet: Various biotin-labeled lectins, such as Dolichos biflorus agglutinin (DBA), Datura stramonium agglutinin (DSA), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Pisum sativum agglutinin (PSA), Sophora japonica agglutinin (SJA), Triticum vulgaris (wheat germ) agglutinin (WGA) were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Immunoprecipitation, SDS Page, Positive Control

Journal: Scientific Reports

Article Title: Shotgun Glycomics Identifies Tumor-Associated Glycan Ligands Bound by an Ovarian Carcinoma-Specific Monoclonal Antibody

doi: 10.1038/s41598-017-15123-z

Figure Lengend Snippet: mAb A4 distinguishes cancerous from non-cancerous ovarian epithelial cell lines. ( A ) Western blotting of IGROV-1 and IOSE523 whole cell lysate shows mAb A4 binding to a multiplicity of targets in IGROV-1, but not in IOSE523 cells. When cell lysates are treated with PNGase-F, binding to IGROV-1 is mostly abolished, indicating that mAb A4 recognizes primarily N-linked glycans. A full-length blot is included as Supplementary Fig. . ( B ) FACS experiments with a panel of plant-derived lectins and sugar-specific antibodies shows that only mAb A4 and peanut agglutinin are able to positively distinguish between cancerous and non-cancerous ovarian epithelial cell lines (N = 3), suggesting that mAb A4 may bind to ovarian cancer-associated glycans. Abbreviations: RCA-I = ricinus communis agglutinin-1, WGA = wheat germ agglutinin, ECL = erythrina cristalli lectin, AAL = aleuria aurantia lectin, BPP = bauhinia purpurea lectin, UEA-I = Ulex Europaeus-1; SBA = soybean agglutinin, PNA = peanut agglutinin, GNL = galanthus nivalis lectin, EBL = elderberry bark lectin/sambucus nigra agglutinin; HPA = helix pomatia agglutinin, VVL = vicia villosa lectin, DBA = dolichol biflorous agglutinin, A4 = mAb A4, α-Lex = anti-Lewis X antibody, α-H = anti-H type I antibody, Neg = negative control. *Represents p < 0.05, Bonferroni corrected Student’s T-test.

Article Snippet: Plant-derived biotinylated lectins were purchased from Vector Laboratories.

Techniques: Western Blot, Binding Assay, Derivative Assay, Negative Control