mouse monoclonal antibody d2-40 acr266b  (Biocare Medical)

Journal: Scientific Reports

Article Title: Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels

doi: 10.1038/s41598-022-26041-0

Figure Lengend Snippet: Effects of cell ratio and vascular endothelial growth factor-C (VEGF-C) concentration on the mesh pattern by lymphatic endothelial cells (LECs) co-cultured with adipose-derived stem cells (ASCs). ( a–l ) Representative images of LECs co-cultured with ASCs and immunostained for podoplanin (a glycoprotein expressed by LECs). The LECs were cultured in medium containing 0 ng/mL VEGF-C ( a – d ), 25 ng/mL VEGF-C ( e – h ) or 50 ng/mL VEGF-C ( i – l ), and the ratio of LECs to ASCs was either 1:4 ( a , b , e , f , i , j ) or 3:2 ( c , d , g , h , k , l ). High-power images of ( a , c , e , g , i , k ) which were skeletonized by ImageJ software to evaluate branches and branch lengths are shown in ( b , d , f , h , j , l ). LECs co-cultured with ASCs at a ratio of 1:4 in 0 ng/mL VEGF-C or 25 ng/mL VEGF-C generated elongated structures but did not form ‘islands’ ( a , b , e , f ). Similar results were obtained when LECs were co-cultured with ASCs at a ratio of 1:4 in 50 ng/mL VEGF-C, although the LEC mesh pattern was denser than that obtained in the lower concentrations of VEGF-C ( i , j ). LECs co-cultured with ASCs at a ratio of 3:2 in 0 ng/mL VEGF-C formed island-like structures with a dense LEC mesh pattern ( c , d ). LECs co-cultured with ASCs at a ratio of 3:2 in 25 ng/mL VEGF-C or 50 ng/mL VEGF-C formed more prominent island-like structures containing a dense and fine LEC mesh pattern ( g , h , k , l ). ( m ) Density of branches in the LEC mesh pattern (number of branches per mm 2 ). Significantly more branches were observed for a LEC/ASC ratio of 3:2 than for a ratio of 1:4 (P < 0.0001, two-way analysis of variance [ANOVA]). However, the concentration of VEGF-C had no significant effect on junction formation (P = 0.83, two-way ANOVA). Pairwise comparisons showed that branch density in the 3:2 ratio/25 ng/mL VEGF-C group was significantly higher than that in the 1:4 ratio/0 ng/mL VEGF-C group or the 1:4 ratio/25 ng/mL VEGF-C group (P < 0.05). All conditions were quantified and compared. Non-significant differences were not denoted on the graphs. The data are presented as the mean ± standard deviation (n = 8 per group). * P < 0.05. ( n ) LEC mesh pattern branch length (mm/mm 2 ). Branch length was longer for a LEC/ASC ratio of 3:2 than for a ratio of 1:4 (P = 0.002, two-way ANOVA). However, VEGF-C had no significant effect on branch length (P = 0.76, two-way ANOVA). All conditions were quantified and compared. Non-significant differences were not denoted on the graphs. The data are presented as the mean ± standard deviation (n = 8 per group).

Article Snippet: Then, the slides were incubated with anti-human podoplanin mouse monoclonal primary antibody (clone D2-40, #413451, Nichirei Biosciences, Japan; 1:10) overnight at 4 °C.

Techniques: Concentration Assay, Cell Culture, Derivative Assay, Software, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels

doi: 10.1038/s41598-022-26041-0

Figure Lengend Snippet: Lymphatic vessel-like structures were evident in a three-layered LEC/ASC sheet (1:4 ratio) 2 weeks after transplantation onto rat gluteal muscle. ( a ) Temperature-responsive culture dish coated with Poly( N -isopropylacrylamide) (PIPAAm), temperature-responsive polymer used to construct cell sheet. At temperature of 37 °C, cells adhere to surface of dish, and temperature decreased to 20 °C enable cells detach from dish preserving their cell–cell junctions, cell surface proteins and extracellular matrix without need of enzymatic treatment which breaks cell–cell junctions. ( b ) Observation of three cell sheets stacked before implantation by stereo microscope. ( c ) Three LEC/ASC sheets were stacked and transplanted onto rat gluteal muscle in vivo, and immunostaining for podoplanin was performed 2 weeks later. The low-power image shows LECs positive for human podoplanin. CS cell sheet. ( d ) High-power image showing a few lymphatic vessel-like structures composed of LECs. ( e ) Positive control image of human podoplanin in human small intestine.

Article Snippet: Then, the slides were incubated with anti-human podoplanin mouse monoclonal primary antibody (clone D2-40, #413451, Nichirei Biosciences, Japan; 1:10) overnight at 4 °C.

Techniques: Transplantation Assay, Polymer, Construct, Preserving, Microscopy, In Vivo, Immunostaining, Positive Control

Journal: Scientific Reports

Article Title: Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels

doi: 10.1038/s41598-022-26041-0

Figure Lengend Snippet: Comparison of lymphatic vessel-like structures between different methods of cell transplantation onto rat gluteal muscle. ( a ) A total of 0.6 × 10 6 LECs and 2.4 × 10 6 ASCs were collected in 100 μL of phosphate-buffered saline for injection. ( b ) In the diffuse network group, three LEC/ASC sheets (1:4 ratio) were stacked for transplantation. ( c ) In the localized network group, two ASC sheets were stacked on one LEC/ASC sheet (3:2 ratio) for transplantation. ( d–f ) Tissue sections immunostained for podoplanin. Nuclei were counterstained with DAPI (blue). The arrowheads show lymphatic vessel-like structures derived from the transplanted lymphatic endothelial cells (LECs). No lymphatic vessel-like structures were observed in the cell injection group; however, host vessels had formed due to inflammation ( d ). ( g–j ) Number ( g ), diameter ( h ), average depth ( i ) and maximum depth ( j ) of lymphatic vessel-like structures staining positively for human podoplanin. The data are presented as the mean ± standard deviation (n = 5 per group). **P < 0.01. ( k–m ) Tissue sections immunostained for human podoplanin (red) and rat LYVE-1 (green). Nuclei were counterstained with DAPI (blue). The arrowheads show lymphatic vessel-like structures derived from the transplanted lymphatic endothelial cells (LECs). No lymphatic vessel-like structures were observed in the cell injection group; however, arrows show appearance of host lymphatic vessels ( k ). Host lymphatic vessels (arrows) and graft lymphatic vessel-like structures (arrowheads) were observed separately in diffuse network group ( l ). Connection of a host LEC with graft lymphatic vessel-like structure was observed in localized network group ( m ).

Article Snippet: Then, the slides were incubated with anti-human podoplanin mouse monoclonal primary antibody (clone D2-40, #413451, Nichirei Biosciences, Japan; 1:10) overnight at 4 °C.

Techniques: Comparison, Transplantation Assay, Saline, Injection, Derivative Assay, Staining, Standard Deviation

Journal: HPB Surgery

Article Title: The Prognostic Significance of Lymphatics in Colorectal Liver Metastases

doi: 10.1155/2014/954604

Figure Lengend Snippet: CD34 and D2-40 expression in colorectal liver metastases. (a) Strong staining of blood vessels using CD34, magnified insert. Arrows indicating CD34 positive blood vessels. (b) Lymphatic vessel staining using D2-40; magnified insert indicating the absence of lymphatic vessels in highly vascular region.

Article Snippet: The sections were immunostained with mouse monoclonal antibody D2-40 used at 0.03475 mg/mL (D2-40, Dako, Victoria, Australia) and LYVE-1 used at 0.0067 mg/mL (Abcam, Cambridge, USA).

Techniques: Expressing, Staining

Journal: HPB Surgery

Article Title: The Prognostic Significance of Lymphatics in Colorectal Liver Metastases

doi: 10.1155/2014/954604

Figure Lengend Snippet: Higher lymphatic vessel density (LVD) in the tumor periphery compared to tumor centre, adjacent, and distal liver. (a) Paraffin embedded section showing lymphatic vessels stained with D2-40 at the tumor periphery and centre, adjacent, and distal liver; magnified inserts of area of interest shown (×400). (b) Enumeration of lymphatic vessel counts revealed higher LVD in the tumor periphery compared to tumor centre, adjacent and distal liver (* P < 0.001).

Article Snippet: The sections were immunostained with mouse monoclonal antibody D2-40 used at 0.03475 mg/mL (D2-40, Dako, Victoria, Australia) and LYVE-1 used at 0.0067 mg/mL (Abcam, Cambridge, USA).

Techniques: Staining

Journal: HPB Surgery

Article Title: The Prognostic Significance of Lymphatics in Colorectal Liver Metastases

doi: 10.1155/2014/954604

Figure Lengend Snippet: LYVE-1 not able to detect lymphangiogenesis in tumor. (a) Paraffin embedded section showing lymphatic vessels stained with D2-40 (×80). (b) Magnified insert highlighting the strong staining of D2-40 expressing lymphatics within the tumor (arrows) (×400). (c) D2-40 did not stain the liver sinusoids or hepatic blood vessels (arrow) (×400). (d) Serial sections stained using immunohistochemistry with LYVE-1; revealed LYVE-1 was not a specific marker for lymphangiogenesis in the liver (×80). (e) LYVE-1 was not able to detect lymphatic vessels in the tumor periphery where D2-40 was able to detect lymphatic vessels (arrows) (×400). (f) LYVE-1 was expressed in liver sinusoids and hepatic blood vessels (arrows) (×400).

Article Snippet: The sections were immunostained with mouse monoclonal antibody D2-40 used at 0.03475 mg/mL (D2-40, Dako, Victoria, Australia) and LYVE-1 used at 0.0067 mg/mL (Abcam, Cambridge, USA).

Techniques: Staining, Expressing, Immunohistochemistry, Marker

Journal: BMC Cancer

Article Title: Coexpression of VEGF-C and COX-2 and its association with lymphangiogenesis in human breast cancer

doi: 10.1186/1471-2407-8-4

Figure Lengend Snippet: (a) : Breast cancer specimen with high vascular endothelial growth factor-C expression. Note the typical cytoplasmatic staining reaction, original magnification 400×. (b) : Breast cancer with high cyclooxygenase-2 expression. Typically granular staining was diffuse in cytoplasm of the cancer cells, original magnification 400×. (c) : D2-40-stained lymphatic vessel (arrows) with tumor cells (T) inside (lymph vessel invasion) was noted, original magnification 200×. (d) : Breast cancer specimen with a high peritumoral lymphatic vessel density (LVD), some of the lymphatic vessels stained for D2-40 are marked with arrows, original magnification 200×. (e-f): Double staining for D2-40 (brown) and Ki-67 (red) of lymph vessels; Positive staining for Ki-67 is seen in nuclei of lymphatic endothelial cells (black arrows), original magnification 400×.

Article Snippet: A monoclonal mouse antihuman D2-40 antibody (Zymed, USA) was used for the staining of lymphatic vessels.

Techniques: Expressing, Staining, Double Staining

Journal: PLoS ONE

Article Title: The Use of Ovarian Cancer Cells from Patients Undergoing Surgery to Generate Primary Cultures Capable of Undergoing Functional Analysis

doi: 10.1371/journal.pone.0090604

Figure Lengend Snippet: Antibodies used for cell characterisation.

Article Snippet: D2-40 , Anti-D2-40 identifies an O-linked sialoglycoprotein present on germ cell tumors but not epithelial cells . Expression was assessed using mouse monoclonal anti-D2-40 antibody and goat anti-mouse Alexafluor 596 secondary antibody , 1∶100 (Primary)) , Dako, Germany.

Techniques: Staining, Marker, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Resection of the mesopancreas (RMP): a new surgical classification of a known anatomical space

doi: 10.1186/1477-7819-5-44

Figure Lengend Snippet: A) . Pancreatic tissue and surrounding vessel-nerve-bundles. Lymphatic vessels stained brown (D2-40). B). Nerve fibres embedded in the fatty tissue of the mesopancreas. Nervesare marked with blue arrows (H&E). C) Mesopancreas, pancreatic tissue in the left upper area. Vessels and nerves (blue arrows) embedded in fatty tissue, lymphatic vessels stained brown (D2-40).D). x = Lymph node in the mesopancreas, lymphatic vessels stained brown. E). Mesopancreas; x = pancreatic tissue, red arrows: nerves; lymphatic vessels stained brown.

Article Snippet: Staining was carried out with Haematoxylin and Eosin (H&E) and the monoclonal antibody D2-40 (mouse anti-human) (Dakocytomation) for the selective representation of lymphatic endothelium and ganglion cells.

Techniques: Staining