Immunohistochemical staining of paraffin embedded human lung with purified ab109189 at a working dilution of 1/1000. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescence staining of A431 cells with purified ab109189 at a working dilution of 1/300, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab109189 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Anti-Thrombomodulin antibody [EPR4051] (ab109189) at 1/10000 dilution (purified) + human placenta lysate at 10 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Flow Cytometry analysis of A431 (human epidermoid carcinoma) cells labeling Thrombomodulin with purified ab109189 at 1/150 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)(1/2000 dilution) was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Unpurified ab109189, at 1/100 dilution, staining Thombomodulin in A431 cells by Immunofluorescence.

ab109189 (purified) at 1/90 immunoprecipitating thrombomodulin in 10 μg human placenta whole cell lysate (Lanes 1 and 2, observed at 100 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

All lanes : Anti-Thrombomodulin antibody [EPR4051] (ab109189) at 1/1000 dilution (unpurified)

Lane 1 : THP-1 cell lysate
Lane 2 : Human placenta lysate
Lane 3 : Human heart lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 60 kDa

Unpurified ab109189, at 1/100 dilution, staining Thrombomodulin in Human placenta tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Unpurified ab109189, at 1/100 dilution, staining Thrombomodulin in Human squamous cervical carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded normal Human spleen tissue using unpurified ab109189 showing +ve staining.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded normal Human tonsil tissue using unpurified ab109189 showing +ve staining.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded normal Human lung tissue using unpurified ab109189 showing +ve staining.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Unpurified ab109189 staining Thrombomodulin in Human artery tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 20% serum for 60 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. A Biotin-conjugated Goatanti-rabbit polyclonal (1/200) was used as the secondary antibody.

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