Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RD (human muscle multinucleated spindle-shaped cell) and HEK-293 (human embryonic kidney epithelial cell) cells labelling heavy chain Myosin/MYH3 with ab325795 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in a part of RD cells and no staining in HEK-293 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : HEK-293 (PMID : 25957469).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-alpha Tubulin mouse monoclonal antibody (ab7291) was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325795 at 1/50 dilution, followed by ab150081 at 1/1000 dilution
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150120 at 1/1000 dilution

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunohistochemical analysis of paraffin-embedded (A) RD (human muscle multinucleated spindle-shaped cell) cell pellet and (B) HEK-293 (Human embryonic kidney epithelial cell) cell pellet tissue labeling heavy chain Myosin/MYH3 with ab325795 at 1/5000 (0.099 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on RD cell pellet (image A), no staining on HEK-293 cell pellet (image B). The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized C2C12 (mouse myoblast) and Differentiated C2C12 cells labelling heavy chain Myosin/MYH3 with ab325795 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in a part of differentiated C2C12 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-alpha Tubulin mouse monoclonal antibody (ab7291) was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunohistochemical analysis of paraffin-embedded Mouse E14.5 embryo tissue tissue labeling heavy chain Myosin/MYH3 with ab325795 at 1/1000 (0.493 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on skeletal muscle of mouse E14.5 embryo tissue (PMID : 36152246). The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

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

Negative control : adult skeletal muscle, adult lung (PMID : 25957469; PMID : 3530499).

Low expression : HEK-293 (PMID : 25957469)

The expression profile observed is consistent with what has been described in the literature (PMID : 3530499, PMID : 12899730, PMID : 31579487, PMID : 25957469 ).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) (1 : 10000) (124KDa).

Exposure time : Lanes 1-4 : 59 seconds; Lanes 5-9 : 15 seconds

All lanes:

Western blot - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (ab325795) at 1/1000 dilution

Lane 1:

Untreated C2C12 (mouse myoblast) whole cell lysate at 20 µg

Lane 2:

C2C12 differentiation to muscle for 3 days whole cell lysate at 20 µg

Lane 3:

RD (human muscle multinucleated spindle-shaped cell) whole cell lysate at 20 µg

Lane 4:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 5:

Mouse E14 forelimb tissue lysate at 20 µg

Lane 6:

Mouse embryo tissue lysate at 20 µg

Lane 7:

Mouse E12.5 embryo tissue lysate at 20 µg

Lane 8:

Mouse adult skeletal muscle tissue lysate at 20 µg

Lane 9:

Mouse adult lung tissue 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 1/20000 dilution

Observed band size: 223 kDa,124 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunohistochemical analysis of paraffin-embedded Mouse adult lung tissue labeling heavy chain Myosin/MYH3 with ab325795 at 1/1000 (0.493 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse adult lung. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunohistochemical analysis of paraffin-embedded Mouse adult skeletal muscle tissue labeling heavy chain Myosin/MYH3 with ab325795 at 1/1000 (0.493 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse adult skeletal muscle. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-heavy chain Myosin/MYH3 antibody [EPR29772-555] (AB325795)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling heavy chain Myosin/MYH3 with ab325795 at 1/5000 (0.099 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human skeletal muscle. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins