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Mouse anti Human podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in human thyroid tissue by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.
Image caption:
Podoplanin expression in human thyroid tissues analyzed by immunohistochemistry.
Immunohistochemistry detection of podoplanin was carried on paraffin-embedded tissue sections. Representative immunostaining results obtained with anti-PDPN monoclonal antibody D2-40. (a) exclusive and strong staining of lymphatic vessels in normal thyroid tissue; (b) follicular adenoma (FA) tissue negative for podoplanin; (c) follicular thyroid carcinoma (FTC) tissue negative for podoplanin; (d) papillary thyroid carcinoma (PTC) case negative for podoplanin; (e-l) intense cytoplasmic staining of PDPN in PTC cells. In e, j and i intense staining of PDPN in tumor cells, with the peritumoral margin negative for podoplanin and strong staining of lymphatic vessels as an internal positive control. Original magnification: a-100x, a-inset -100x; b-200x, b-inset -100x; c-200x; d-200x, e-100x, f-100x, g-200x; h-200x, i-100x, j-200x, k-200x, l-400x. Of 112 PTC cases, 45 (40%) displayed ectopic podoplanin expression in the cancer cells.

From: Rudzińska M, Gawel D, Sikorska J, Karpińska KM, Kiedrowski M, Stepień T, et al. (2014) The Role of Podoplanin in the Biology of Differentiated Thyroid Cancers.
PLoS ONE 9(5): e96541.

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Mouse anti Human podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in human thyroid cancer derived cell lines by western blotting and immunofluorescence.
Image caption:
Podoplanin transcript and protein expression levels in differentiated thyroid cancer derived cell lines. A. PDPN mRNA expression in human thyroid cancer cell lines. RT-qPCR was used to evaluate levels of the transcript encoding podoplanin in total RNA prepared from NTHY control cells, PTC-derived TPC1 and BcPAP cell lines, and FTC-derived FTC133 and CGTH-W-1 cell lines. Data from four independent experiments performed in triplicate are expressed as the mean ±SEM, **P<0.001. B. Podoplanin protein levels in human thyroid cancer cell lines. Protein extracts (30 μg) were subjected to Western blot analysis with anti-PDPN monoclonal antibody D2-40 and β-actin antibody as a loading control. C. Immunofluorescence analysis of the podoplanin expression in TPC1, BcPAP, FTC133 and CGTH-W-1 thyroid cancer cell lines. PDPN was visualized by staining using anti-PDPN monoclonal antibody D2-40 followed by DyLight549-conjugated secondary antibody (red), and nuclei were counterstained with DAPI (blue). Magnification 1000x.

From: Rudzińska M, Gawel D, Sikorska J, Karpińska KM, Kiedrowski M, Stepień T, et al. (2014) The Role of Podoplanin in the Biology of Differentiated Thyroid Cancers.
PLoS ONE 9(5): e96541.

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Mouse anti Human podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in TCP1 cells before and following podoplanin silencing with siRNA by western blotting and immunofluorescence.
Image caption:
Down-regulation of podoplanin expression in TPC1 cells following transfection with PDPN-specific siRNA. A. RT-qPCR analysis of PDPN mRNA levels in TPC1 thyroid cancer cells 48 h after transfection with 30 nM siRNA specific for PDPN (siPDPN) or a negative control siRNA (siNEG). The results were normalized to the 18S rRNA level and bars represent the average fold change in PDPN transcript abundance in cells transfected with siPDPN compared with cells transfected with siNEG. The results are representative of four independent experiments. Data are presented as the mean ±SEM, **P<0.001. B. Western blot analysis of podoplanin and β-actin proteins in TPC1 cells before (0 h) and 48 h after transfection with siPDPN and control siNEG. C. Immunofluorescence staining of podoplanin protein in TPC1 cells transfected with siPDPN and control siNEG. Cells were stained with anti-PDPN monoclonal antibody D2-40 followed by DyLight549-conjugated secondary antibody (red), and counterstained with DAPI (blue). Magnification 1000x. D. Effect of PDPN on cell viability. D, left panel. Proliferation was measured at 24 and 48 h after transfection of TPC1 cells with siPDPN or control siNEG. Cells seeded in 96-well plates were treated with XTT mixture reagent and formazan formation was measured at 450 nm to determine the number of viable cells. Data are expressed as the mean ±SEM of at least three independent experiments performed in quintuplicate. D, right panel. Apoptosis was measured at 48 h after transfection of TPC1 cells with siPDPN or control siNEG. Cells were collected and stained with FITC Annexin V and propidium iodide, followed by flow cytometry. Representative measurements of the percentage of Annexin V+ cells are presented. Each bar represents the mean ±SEM of at least three independent experiments performed in quadruplicate. E. Function of podoplanin in cell adhesion. TPC1 cells transfected with siPDPN display adhesion capacity comparable to those of siNEG-transfected cells. Briefly, 8000 transfected cells were seeded in the wells of 96-well plates. After incubation for 24 or 48 h, the cell monolayers were washed, fixed with 4% formaldehyde for 15 min and stained with crystal violet (Merck, USA). The stained cells were lysed by treatment with 2% SDS, then the intensity of the released stain was quantified by spectrophotometry at 550 nm using a Labsystems Multiscan RC microplate reader (Thermo Fisher Scientific, Canada). Data represents three separate experiments.

From: Rudzińska M, Gawel D, Sikorska J, Karpińska KM, Kiedrowski M, Stepień T, et al. (2014) The Role of Podoplanin in the Biology of Differentiated Thyroid Cancers.
PLoS ONE 9(5): e96541.

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Mouse anti Human Podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in human synovial tissue by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.
Image caption:
Podoplanin is expressed in human synovial tissue in RA. Immunohistochemistry (IHC) of human synovial tissue from (A-D, I) rheumatoid arthritis (RA), (E-H, J) osteoarthritis (OA) (A-D, E-H, J) using antipodoplanin antibody (D2-40) or (I) mouse immunoglobulin G1. (J) Positive control showing lymph vessels (arrow). (K) IHC staining score of podoplanin on human synovial tissue from 18 RA and 9 OA patients. Double immunofluorescence staining of (L and M) RA and (N) OA synovium using antipodoplanin (green) and anti-cadherin-11 (red) antibodies. Note the extensive hyperplasia of the podoplanin-positive lining layer cells (C and D, L) and the podoplanin-positive lymph vessel (arrowhead in N) but negative lining layer in OA (N). L, lumen. ***statistical significance P < 0,0001.

From: Ekwall AK, Eisler T, Anderberg C, Jin C, Karlsson N, Brisslert M, Bokarewa MI. The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis. Arthritis Res Ther. 2011 Mar 7;13(2):R40.

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Mouse anti Human Podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in human synovial tissue by western blotting.
Image caption:
Anti-podoplanin antibody D2-40 recognizes 45kD band in Western blot of synovial protein extracts. Western blot of extracted membrane proteins from human synovial tissue from a patient with rheumatoid arthritis (RA) (lane 1), cell pellet of human MRC-5 lung fibroblast cell line (lane 2) and recombinant immature podoplanin core protein (lane 3) separated on a 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel probed with the antipodoplanin antibody (D2-40).

From: Ekwall AK, Eisler T, Anderberg C, Jin C, Karlsson N, Brisslert M, Bokarewa MI. The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis. Arthritis Res Ther. 2011 Mar 7;13(2):R40.

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Mouse anti Human Podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin in human synovial tissue by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.
Image caption:
Podoplanin is expressed in FLS in areas of synovial transformation. Immunohistochemistry of (A and B) frozen and (C-J) paraffin-embedded human synovial tissue from patients with rheumatoid arthritis (RA) using antibodies against (A and C) podoplanin (D2-40), (B) CD90 (AS02) and (D) cadherin-11 (3B2H5) (arrowhead). Double immunofluorescence staining analysed by confocal microscopy showing, in green, (E) podoplanin 18H5 and (F-J) podoplanin D2-40, and in red, (E) cadherin-11 (3B2H5), (F) CD68 (KP1), (G and overview in H) E-cadherin (H-108), (I) a-smooth muscle actin (a-sma) (PA1-37024) and (J) matrix metalloproteinase 9 (MMP-9). (E) Note colocalization of podoplanin and cadherin-11 (arrowhead). L, lumen.

From: Ekwall AK, Eisler T, Anderberg C, Jin C, Karlsson N, Brisslert M, Bokarewa MI. The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis. Arthritis Res Ther. 2011 Mar 7;13(2):R40.

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Mouse anti Human Podoplanin antibody, clone D2-40 (MCA2543) used for the detection of podoplanin rheumatoid arthritis fibroblast-like synoviocytes by immunocytochemistry, immunofluorescence and flow cytometry
Image caption:
Podoplanin is expressed in cultured primary FLS and the expression is increased by pro-inflammatory cytokines. (A) Immunofluorescence staining of primary rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) showing podoplanin (red) and actin stress fibres (green). Note accumulated podoplanin staining in membrane protrusions (arrowheads). (B) Magnification of podoplanin-positive RA-FLS. (C and D) Flow cytometry (FACS) of primary FLS cultures from patients with RA (filled bars) and patients with osteoarthritis (OA) (striped bars) showing the percentage of positive cells of viable cell populations using (C) antipodoplanin and phenotype markers (CD90, CD68 and CD29) and (D) cadherin-11 antibodies. (E) Immunocytochemistry of an aggressively growing RA-FLS culture using antipodoplanin antibody. Note the dendritic phenotype with long cytoplasmatic protrusions. (F) Representative flow cytometry plot of primary RA-FLS stained for podoplanin and α-smooth muscle actin (α-sma) showing the double-positive population (podoplanin+ and α-sma+) of 58.2% in the upper right quadrant. (G) Graph showing the percentage of podoplanin-positive primary FLS by flow cytometry at baseline, 24 and 48 hours of stimulation with control (complete medium) (open circles), 10 ng/ml tumour necrosis factor (TNF)-α (filled squares), 1 ng/ml interleukin (IL)-1β (open diamond) and 1 ng/ml transforming growth factor β receptor 1 (TGF-β1) (filled triangles), respectively, of a representative culture. The experiment was run in triplicate and repeated four times using different OA-FLS and RA-FLS cultures, which showed similar results but starting at different baseline levels of podoplanin expression.

From: Ekwall AK, Eisler T, Anderberg C, Jin C, Karlsson N, Brisslert M, Bokarewa MI. The tumour-associated glycoprotein podoplanin is expressed in fibroblast-like synoviocytes of the hyperplastic synovial lining layer in rheumatoid arthritis. Arthritis Res Ther. 2011 Mar 7;13(2):R40.