All lanes : Anti-Actin antibody [EPR16769] (ab179467) at 1/20000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate
Lane 5 : Rat brain tissue lysate
Lane 6 : Rat heart tissue lysate
Lane 7 : Rat kidney tissue lysate
Lane 8 : Rat spleen tissue lysate
Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human prostate hyperplasia tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining on smooth muscle cells is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling Actin with Purified ab179467 at 1:100 dilution (6.98 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelia cell) cells labeling Actin with Purified ab179467 at 1:100 dilution ( 6.98 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling Actin with ab179467 at 1/50 dilution, followed by Goat anti-rabbit Alexa Fluor^® 488 (IgG) (ab150077) secondary antibody at 1/200 dilution (green). Cytoplasm staining on NIH/3T3 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution (red).

The negative controls are as follows;
1. ab179467 at 1/50 dilution followed by ab150120 (goat anti-mouse Alexa Fluor^® 594 secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (goat anti-rabbit Alexa Fluor^® 488 (IgG H&L) at 1/200 dilution.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling Actin with purified ab179467 at 1/70 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

All lanes : Anti-Actin antibody [EPR16769] (ab179467) at 1/20000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : 293T (Human epithelial cells from embryonic kidney) whole cell lysates
Lane 3 : Human skeletal muscle tissue lysate
Lane 4 : Human fetal spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Actin antibody [EPR16769] (ab179467) at 1/5000 dilution

Lane 1 : Human fetal brain tissue lysate
Lane 2 : Human fetal heart tissue lysate
Lane 3 : Human fetal kidney tissue lysate
Lane 4 : Human fetal spleen tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Actin antibody [EPR16769] (ab179467) at 1/20000 dilution

Lane 1 : UMNSAH/DF-1 (Transformed chicken embyronic fibroblast cells) whole cell lysates)
Lane 2 : Human cardiac muscle tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling Actin with ab179467 at 1/1000 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Cytoplasm staining is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary ab, secondary ab is prediluted HRP Polymer for Rabbit/Mouse IgG.

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

Actin was immunoprecipitated from 1mg of NIH/3T3 (Mouse embyro fibroblast cells) whole cell extract with ab179467 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab179467 at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as secondary antibody at 1/1000 dilution.

Lane 1: NIH/3T3 whole cell extract.
Lane 2: PBS instead of NIH/3T3 whole cell extract.

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