Immunocytochemistry/ Immunofluorescence - Caveolin 1 antibody - Caveolae Marker (ab18199)

ICC/IF image of ab18199 stained NIH/3T3 cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18199, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Western blot - Caveolin 1 antibody - Caveolae Marker (ab18199)

All lanes : Anti-Caveolin 1 antibody - Caveolae Marker (ab18199) at 1 µg/ml

Lane 1 : Mouse Heart Lysate
Lane 2 : Human Heart Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 20 kDa
Observed band size : 20 kDa

Western blot - Caveolin 1 antibody - Caveolae Marker (ab18199)

All lanes : Anti-Caveolin 1 antibody - Caveolae Marker (ab18199) at 1 µg/ml

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Heart (Mouse) Tissue Lysate
Lane 4 : Heart (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 20 kDa
Observed band size : 20 kDa

Immunocytochemistry/ Immunofluorescence - Caveolin 1 antibody - Caveolae Marker (ab18199)

ab18199 staining human HT1080 cells by ICC/IF.  Cells were PFA fixed and permeabilized in Triton X-100 prior to incubating with ab18199 at 10 µg/ml for 1 hour at 20°C.  A Texas Red® conjugated donkey anti-rabbit antibody was used as the secondary.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Caveolin 1 antibody - Caveolae Marker (ab18199)

ICC/IF image of ab18199 stained MEF7cells. The cells were 4% PFA 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 (ab18199, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.

Immunoprecipitation - Anti-Caveolin 1 antibody - Caveolae Marker (ab18199)

Caveolin 1 - Caveolae Marker was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to Caveolin 1 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, Mouse Brain whole tissue lysate 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18199.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 20kDa: Caveolin 1 - Caveolae Marker.