Western blot - Actin antibody [ACTN05 (C4)] (ab3280)

Predicted band size : 42 kDa

Lanes: 1, 4, 7 - NIH3T3; 2, 5, 8 - MDA MB 321; 3, 6, 9 - HeLa

Lanes: 1-3: 4^oC (1 freeze/thaw);  4-6: 4^oC (2 freeze/thaws); 7-9 :   4^oC (4 freeze/thaws)   

Western blot - Actin antibody [ACTN05 (C4)] (ab3280)

All lanes : Anti-Actin antibody [ACTN05 (C4)] (ab3280) at 1/2000 dilution

Lane 1 : Mouse liver whole tissue lysate (mouse 1).
Lane 2 : Mouse liver whole tissue lysate (mouse 2).
Lane 3 : Mouse liver whole tissue lysate (mouse 3).

Lysates/proteins at 50 µg per lane.

Secondary
An HRP-conjugated goat anti-mouse IgG polyclonal. at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 42 kDa


Blocking Step: 5% Milk for 30 minutes at 25°C.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Frozen sections) - Actin antibody [ACTN05 (C4)] (ab3280)

ab3280 staining Actin in mouse heart tissue sections by IHC-Fr (formaldehyde-fixed frozen sections). Tissue samples were fixed with formaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% serum for 1 hour at 25ºC. The sample was incubated with primary antibody (1/250 in PBS-Tween) at 4ºC for 12 hours. An Alexa Fluor^®488-conjugated Goat polyclonal to mouse IgG (1/250) was used as secondary antibody. Nuclei were stained with DAPI.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Actin antibody [ACTN05 (C4)] (ab3280)

ab3280 (1µg/ml) staining actin in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining throughout the tissue.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Immunocytochemistry/ Immunofluorescence - Actin antibody [ACTN05 (C4)] (ab3280)

ICC/IF image of ab3280 stained Hepp cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3280, 10µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Immunocytochemistry/ Immunofluorescence - Actin antibody [ACTN05 (C4)] (ab3280)

ICC/IF image of ab3280 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3280, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.