Recombinant Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500)

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3(Mouse embryonic fibroblast) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/500 dilution (5.2 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Alexa Fluor^® 488 (ab197240) and Alexa Fluor^® 647 (ab196919) conjugated versions are available for this clone.

FoxA1 (red) and alpha smooth muscle actin (green) staining are shown by indirect immunofluorescence on sections of prostate from mice of the indicated genotypes.

Wild type: 21 weeks, Tgfbr2^r/r: 44 weeks, Pten^r/r: 21 weeks, Pten^r/r;Tgfbr2^r/r: 11 weeks, Apc^r/r: 36 weeks, and Apc^r/r;Tgfbr2^r/r: 24 weeks old.

IF images were captured on an Olympus BX51 microscope and DP70 digital camera, or on a Nikon Eclipse NI-U and captured with a DS-QI1 camera with NIS Elements software.

 

All lanes : Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575) at 1/5000 dilution (purified)

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

Blocking and dilution buffer: 5% NFDM/TBST

Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/20 (red). 

Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies. 

Alexa Fluorr^®488 (ab197240) and Alexa Fluorr^®647 (ab196919) conjugated versions are available for this clone. 

 

 

Immunocytochemistry/ Immunofluorescence analysis of C6(Rat glial tumor glial cell) cells labeling alpha smooth muscle Actin with purified ab32575 at 1/100 dilution (0.71 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Different batches of ab32575 were tested on A431 (Human epidermoid carcinoma epithelial cell) lysate at 0.005 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 42 kDa.

All lanes : Anti-alpha smooth muscle Actin (acetyl E3) + ACTG2 (acetyl E3) antibody [E184] (ab32575) at 1/5000 dilution (purified)

Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 42 kDa
Observed band size: 42 kDa

Blocking and dilution buffer: 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labeling alpha smooth muscle Actin (green) with purified ab32575 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counterstained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 Alexa Fluor^®594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).  

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse smooth muscle tissue labeling alpha smooth muscle Actin with purified ab32575 at a dilution of 1/200. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500).

Negative control using PBS instead of primary antibody.

Counterstained with hematoxylin.

Following reconstitution in PBS with 10% DMSO, peptides (1 ug per mL) were immobilised in PBS on an ELISA plate overnight. After blocking in 5% BSA, primary antibody (ab271180) was added in a concentration range of 0.017-1000 ng per mL.

Pre-adsorbed secondary antibody goat anti-rabbit IgG H&L (HRP, ab97080) was used at 1/20000 dilution.

Following reconstitution in PBS with 10% DMSO, peptides (1 ug per mL) were immobilised in PBS on an ELISA plate overnight. After blocking in 5% BSA, primary antibody (ab271180) was added in a concentration range of 0.017-1000 ng per mL.

Pre-adsorbed secondary antibody goat anti-rabbit IgG H&L (HRP, ab97080) was used at 1/20000 dilution.