Anti-Caveolin-1抗体- Caveolae Marker (ab2910)

ab2910 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (ab255371) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab2910 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Caveolin 1 was immunoprecipitated using 5 µg of ab2910 from lysate of Mouse Heart (Lane 3) using the Dynabeads^® Protein A Immunoprecipitation Kit. Normal Rabbit IgG was used as a Isotype control (Lane 2). 10 % input represents the cell extract used for immunoprecipitation (Lane 1). Western blot analysis was performed using Caveolin 1 Rabbit Polyclonal Antibody and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

The specificity of ab2910 was demonstrated by CRISPR targeted CAV1 knockout in HeLa cells. Western blot analysis of whole cell lysates using this antibody showed no detection of caveolin 1 protein expression in knockout cells compared to the protein detected at ~22kDa in wild-type HeLa cells. 

Western blot analysis was performed on whole cell extracts (20 µg lysate). The blots were probed with Anti-ab2910 (1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. A 17 kDa band corresponding to Caveolin-1 was observed across cell lines and tissues tested. Known quantity of protein samples were electrophoresed using Novex^® NuPAGE^® 12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot^® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

Immunofluorescence analysis of Caveolin 1 was done on 70% confluent log phase A-375 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ab2910 at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 488 conjugate at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor^® 555 Rhodamine Phalloidin. Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Rat astrocytes stained with fluorescently labeled Caveolin-1 antibody.

Primary antibody is ab2910 at a dilution of 1/500 and the secondary antibody is Texas red labeled anti-rabbit IgG at a dilution of 1/1000.

This image was kindly supplied as part of the review submitted by Donghui Zhu.

ab2910 staining Caveolin-1 - Caveolae Marker in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde. Samples were incubated with primary antibody (1/200 in PBS + 0.05% Saponin) for 1 hour at 37°C. A Cy3^®-conjugated Donkey anti-rabbit polyclonal (1/500) was used as the secondary antibody.

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