Anti-Podoplanin antibody [EPR22182]

• Flow Cyt

Lab

Flow Cytometry - Anti-Podoplanin antibody [EPR22182] (AB236529)

Flow cytometry overlay histogram showing left U-87 MG positive cells and right negative HUVEC stained with ab236529 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab236529) (1x 106 in 100μl at 1.0 μg/ml (1/500)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Podoplanin antibody [EPR22182] (AB236529)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized U-2 OS (human bone osteosarcoma epithelial cell line) cell line labeling Podoplanin with ab236529 at 1/300 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Podoplanin antibody [EPR22182] (AB236529)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Podoplanin with ab236529 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in lymphatic endothelial cells of human colon (PMID : 28101903; PMID : 27798887) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Podoplanin antibody [EPR22182] (AB236529)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized U-87 MG (humanglioblastoma-astrocytoma epithelial cell line) cell line labeling Podoplanin with ab236529 at 1/300 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Podoplanin antibody [EPR22182] (AB236529)

Fluorescence multiplex immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded Human lung tissue. Panel A : Merged staining of anti-Podoplanin / gp36 (magenta; Opal™690), anti-Surfactant protein D/SP-D (green; Opal™520) and anti-Sialoadhesin/CD169 (red; Opal™570) on human lung. Panel B : Anti-Podoplanin / gp36 stained on alveolar type I cells. Panel C : Anti-Surfactant protein D/SP-D stained on alveolar type II cells. Panel D : Anti-Sialoadhesin/CD169 stained on macrophages. The section was incubated in three rounds of staining : in the order of ab236529 and ab234257 for 30 mins, then ab245735 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Counterstained with DAPI.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Podoplanin antibody [EPR22182] (AB236529)

Immunohistochemical analysis of paraffin-embedded human mesothelioma tissue labeling Podoplanin with ab236529 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining in tumor cells of human mesothelioma (PMID : 16670463) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-Podoplanin antibody [EPR22182] (AB236529)

Podoplanin was immunoprecipitated from 0.35 mg of U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate with ab236529 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab236529 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : U-2 OS whole cell lysate 10 μg (Input).

Lane 2 : ab236529 IP in U-2 OS whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab236529 in U-2 OS whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-Podoplanin antibody [EPR22182] (ab236529)

Predicted band size: 16 kDa

false

• WB

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Western blot - Anti-Podoplanin antibody [EPR22182] (AB236529)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID : 10408868; PMID : 17279584).

Negative control : HUVEC (PMID : 21920451).

All lanes:

Western blot - Anti-Podoplanin antibody [EPR22182] (ab236529) at 1/1000 dilution

Lane 1:

U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 16 kDa

Observed band size: 38 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-Podoplanin antibody [EPR22182] (AB236529)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID : 10408868; PMID : 17279584).

All lanes:

Western blot - Anti-Podoplanin antibody [EPR22182] (ab236529) at 1/1000 dilution

Lane 1:

Human seminoma lysate at 20 µg

Lane 2:

Human tonsil lysate at 20 µg

Lane 3:

Human lymph node lysate at 20 µg

Lane 4:

Human stomach lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 16 kDa

Observed band size: 38 kDa

false

Exposure time: 3min

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Podoplanin antibody [EPR22182] (AB236529)

Immunohistochemical analysis of paraffin-embedded human Kaposi's sarcoma tissue labeling Podoplanin with ab236529 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and cytoplasmic staining in tumor cells of human Kaposi's sarcoma (PMID : 11950918) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.