Immunocytochemistry/ Immunofluorescence - Vinculin antibody [SPM227] (ab18058)

ab18058 at 1/200 staining Chinese Hamster Ovary cells by ICC/IF. The cells were permeabilized with 100% methanol and then blocked with 1% BSA/ 4% goat serum prior to incubation with the antibody for 30 minutes. A rhodamine conjugated goat antibody was used as the secondary.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Vinculin antibody [SPM227] (ab18058)

ab18058 staining mouse 3T3 cells by ICC/IF.  Cells were PFA fixed and permeabilized in Triton X-100 prior to blocking in 1% BSA for 30 minutes at 25°C.  The primary antibody was diluted 1/100 and incubated with the sample for 1 hour at 25°C, ab6785, diluted 1/100, was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Mr John Maloney

Western blot - Vinculin antibody [SPM227] (ab18058)

Anti-Vinculin antibody [SPM227] (ab18058) at 1/1000 dilution + Whole cell lysate prepared from human lung cells at 40 µg

Secondary
HRP conjugated goat polyclonal to mouse IgG at 1/1000 dilution

Predicted band size : 130 kDa
Observed band size : 117 kDa (why is the actual band size different from the predicted?)


The image is a courtsey of an anonymous abreview.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-Vinculin antibody [SPM227](ab18058)

Ab18058 staining human normal prostate. Staining is localised to the nucleus and cytoplasm.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, 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 we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Western blot - Vinculin antibody [SPM227] (ab18058)

Predicted band size : 130 kDa

Lanes: 1, 4, 7 - Hamster Lung; 2, 5, 8 - K562; 3, 6, 9 - CHO

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

Immunocytochemistry/ Immunofluorescence - Vinculin antibody [SPM227] (ab18058)

ICC/IF image of ab18058 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 (ab18058, 10µg/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.