Anti-Villin 抗体 [SP145] - BSA and Azide free (ab245749)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab130751).

Flow cytometry overlay histogram showing left HT-29 positive cells and right negative PANC-1 stained with ab130751 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab130751) (1x 10⁶ in 100μl at 0.2μg/ml (1/11200)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HT-29 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A: merged staining of anti-Villin (ab245749, gray; Opal™690), anti-liver FABP (ab240401, green; Opal™520) and anti-MUC2 (ab272692, red; Opal™570) on human colon. Panel B: anti-liver FABP stained on enterocytes. Panel C: anti-Villin stained on apical border. Panel D: anti-MUC2 stained on goblet cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining: in the order of ab245749 (1/1000 dilution), ab240401 (1/8000 dilution), and ab272692 (1/5000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab130751).

ab130751 staining Villin in mouse intestine tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in 10mM citrate buffer, pH 6. Samples were incubated with primary antibody (1/1000) for 16 hours at 4°C. An Alexa Fluor® 568-conjugated goat anti-rabbit IgG monoclonal (1/250) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab130751).

Overlay histogram showing Caco 2 cells stained with ab130751 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab130751, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab130751). 

Villin was immunoprecipitated using 0.5mg SW480 whole cell extract, 5µg of Rabbit polyclonal to Villin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, SW480 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab130751.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 93kDa; Villin

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab130751).

ab130751, at 1/100 dilution, staining Villin in formalin-fixed, paraffin-embedded Human colon tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab130751).