Recombinant Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labelling Caveolin-1 with purified ab32577 at 1/2000 dilution (0.06 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse lung tissue sections labelling Caveolin-1 with purified ab32577 at 1/2000 dilution (0.06 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue sections labelling Caveolin-1 with purified ab32577 at 1/2000 dilution (0.06 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Lanes 1 - 4: Merged signal (red and green). Green - ab32577 observed at 20 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab32577 was shown to react with Caveolin-1 in wild-type A431 cells in western blot. Loss of signal was observed when CAV1 knockout sample was used. A431 wild-type and CAV1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab32577 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab32577 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 ab32577 at 1/200 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).

Flow cytometric analysis of 4% Paraformaldehyde fixed 90% Methanol permeabilized A431 (Human epidermoid carcinoma epithelial cell) cells labelling Caveolin-1 with ab32577 at 1/20 dilution (10 µg/ml) compared with a Rabbit monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 was used as the secondary antibody.

Lanes 1 - 4: Merged signal (red and green). Green - ab32577 observed at 20 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab32577 was shown to react with Caveolin-1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255371 (knockout cell lysate ab263806) was used. Wild-type and Caveolin-1 knockout samples were subjected to SDS-PAGE. ab32577 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

The two isoforms of Caveolin-1 have been described in the literatures (PMID: 12816877, 11748292 and 14992406). We are unsure about the nature of the 250kDa bands. 

The two isoforms of Caveolin-1 have been described in the literatures (PMID: 12816877, 11748292 and 14992406). We are unsure about the nature of the 250kDa bands.

Immunocytochemistry analysis of Jurkat (Human lung carcinoma epithelial cell) cells labeling Caveolin-1 with purified ab32577 at 1/50 dilution (2.3 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150078) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.