Western blot - VCP antibody [5] (ab11433)

Western blot of VCP from CA46 cell lysate using ab11433.

Immunocytochemistry/ Immunofluorescence - VCP antibody [5] (ab11433)

ab11433 staining VCP in Human H1299 cells by Immunocytochemistry/ Immunofluorescence. The cells were paraformaldehyde fixed, permeabilized in 0.1% Triton X-100  and were blocked in 10% serum at 25°C for 1 hour. Primary antibody was used at 1/1000 dilution (PBS) and incubated with sample for 1 hour at 37°C. An Alexa Fluor^® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/1000 dilution. Image shows green color staining with ab11433 and nuclei were stained blue with DAPI.    

This image is courtesy of an anonymous Abreview.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-VCP antibody [5](ab11433)

ab11433 (1µg/ml) staining VCP in human left ventricle using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear, cytoplasmic and cell membrane staining of cardiomyocutes and smooth muscle cells of cardiac artery.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 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.

Flow Cytometry - VCP antibody [5] (ab11433)

ab24762 detecting VCP in Human platelets by Flow Cytometry. Platelets were isolated by spinning Platelet rich plasma on Histopaque. Cells were fixed in PFA and permeabilized in 0.1% Triton-X100 in 2% BSA for 30 minutes. The primary antibody was diluted 1/400 in 2% BSA in PBS and incubated with the sample for 18 hours at 4ºC. An Alexa Fluor® 488-conjugated Goat anti-Mouse IgG (H+L) antibody, diluted 1/500 was used as the secondary antibody.

Abbreviations in the figure:
P : Permeabilized;
US Unstained, Red Peak;
IgG Rb: IgG Mouse (Isotype Control), Blue Peak;
VCP: Green Peak.

This image is courtesy of an Abreview by Mahesh Shivananjappa.

Western blot - Anti-VCP antibody [5] (ab11433)

Anti-VCP antibody [5] (ab11433) at 1/2000 dilution + whole cell lysate prepared from mouse embryonic fibroblasts at 20 µg

Secondary
Alexa-Fluor 680 conjugated goat anti-mouse polyclonal at 1/10000 dilution

Observed band size : 97 kDa (why is the actual band size different from the predicted?)

Image courtesy of an anonymous Abreview.