Immunohistochemical analysis of paraffin-embedded on human skeletal muscle labeling Desmin with ab15200 at 1/200 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). Counter stained with hematoxylin.

Immunofluorescence staining of Desmin using ab15200 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab15200 at 0.1 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab15200 gave comparable results using MeOH fixation (100%, 5 min).

Immunocytochemistry/Immunofluorescence analysis of C2C12 (mouse muscle) cells labelling Desmin with ab15200 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

ab15200 staining rat differentiated skeletal muscle cells by ICC/IF.

Cultured differentiated skeletal muscle cells were harvested from Lewis rats, 2% paraformaldehyde treated for 15 min to fix the cells, and then permealized with Triton-X100 (0.1%) for 10 min. The cells were then incubated with ab15200 at 1/200 overnight at 4°C. The image was taken with a confocal laser scanning microscope and shows desmin expressing skeletal myocytes (green-cytoplasmic localization). Note that desmin forms short thickened filamentous structures in the cell cytoplasm, along with prominent spot-like cytoplasmic aggregates that are composed of densely packed filaments.

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Immunocytochemistry/Immunofluorescence analysis of RMS13 (human rhabdomyosarcoma) cells labelling Desmin with ab15200 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells (negative cell line) showing weak staining of Desmin with ab15200 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

ab15200 staining Desmin in rat Smooth muscle cells from mesenteric artery by Immunocytochemistry/ Immunofluorescence. Cells were fixed with 4% paraformaldehyde in PSS for 4 min at 4°C and permeabilized with 0.3% Triton x100 before blocking with 2% BSA was done for 30 minutes at 20°C. Samples were incubated with primary antibody (1/300 in PSS with 2%BSA and 0.3% Triton X-100) for 14 hours at 4°C. An MFP 555-conjugated donkey polyclonal to rabbit IgG was used as secondary antibody at 1/400 dilution.

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Immunohistochemical analysis of paraffin-embedded on human cardiac muscle labeling Desmin with ab15200 at 1/200 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). Counter stained with hematoxylin.

Immunohistochemical analysis of paraffin-embedded on rat cardiac muscle labeling Desmin with ab15200 at 1/200 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). Counter stained with hematoxylin.

ab15200 staining Desmin in Mouse skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at room temperature; antigen retrieval was enzymatic. Samples were incubated with primary antibody (1/800 in blocking buffer) for 16 hours at 4°C. An undiluted HRP-conjugated Horse anti-rabbit IgG polyclonal was used as the secondary antibody.

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Ab15200 positively staining desmin in formaldehyde fixed mouse heart tissue (1/200). Ab15200 was used in conjunction with goat anti rabbit (biotin).

The reviewer reported no difference in the quality of staining obtained when enzymatic epitope unmasking was preformed.

This image is an edited version of an image submitted courtesy of an Abreview by Khaled Chatila on 11 October 2005. For further information relating to the protocol please refer to the Abreview.

Immunohistochemical analysis of PFA-fixed paraffin-embedded murine nephric tissue, labelling Desmin with ab15200 at a dilution of 1/250 incubated for 18 hours at 4°C. Heat mediated antigen retrival was with citrate buffer. Blocking was with Dako serum-free protein block at 100% incubated for 1 hour at 23°C. Secondary was a donkey anti-rabbit polyclonal HRP conjugate at 1/200.

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Anti-Desmin antibody - Cytoskeleton Marker (ab15200) at 1 µg/ml + Human placenta tissue lysate - total protein (ab29745) at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 52 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?
Additional bands at: 72 kDa (possible non-specific binding)