All lanes : Anti-PAI1 antibody (ab66705) at 1 µg/ml

Lane 1 : HUVEC (Human Umbilical Vein Endothelial Cell) Whole Cell Lysate
Lane 2 : Human Aortic Endothelial Cell Lysate (HAEC)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 45 kDa


Exposure time: 4 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab66705 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes : Anti-PAI1 antibody (ab66705) at 1 µg/ml

Lane 1 : HUVEC (Human Umbilical Vein Endothelial Cell) Whole Cell Lysate
Lane 2 : Human Aortic Endothelial Cell Lysate (HAEC)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 42,45 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab66705 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

ab66705 staining PAI1 in MCF-7 cells treated with splitomicin (ab141120), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of splitomicin, as described in literature.
The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab141120 (splitomicin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue

IHC image of PAI1 staining in Human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab66705, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of PAI1 staining in Mouse xenograft tumor formalin fixed paraffin embedded tissue section. The section was pre-treated using heat mediated antigen retrieval with 10 mM citrate buffer (pH6). The section was then incubated with ab66705, 1/1000, for 16 hours at 4ºC and detected using undiluted Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880). 

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ab66705 staining PAI1 in HeLa cells. The cells were fixed with 100% methanol (5 min), 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 the antibody ab66705 at 1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

ab66705 staining PAI1 in HeLa cells treated with dynasore (ab120192), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of dynasore, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120192 (dynasore) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with Dyngo-4a™ (ab120689), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of Dyngo-4a™, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab120689 (Dyngo-4a™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with dynole-34-2™ (ab120463), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of dynole-34-2™, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120463 (dynole-34-2™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with iminodyn-22™ (ab120461), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of iminodyn-22™, as described in literature.
The cells were incubated at 37°C for 48h in media containing different concentrations of ab120461 (iminodyn-22™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with MiTMAB™ (ab120466), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of MiTMAB™, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab12046 (MiTMAB™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with OcTMAB™ (ab120467), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of OcTMAB™, as described in literature.
The cells were incubated at 37°C for 24h in media containing different concentrations of ab120467 (OcTMAB™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HeLa cells treated with dynole-31-2™ (ab120464), by ICC/IF. No change in PAI1 expression with increased concentration of dynole-31-2™ (negative control for dynole 34-2™ (ab120463), as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of (ab120464 (dynole-31-2™) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HepG2 cells treated with BAPTA sodium salt (ab120449), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of BAPTA sodium salt, as described in literature.
The cells were incubated at 37°C for 4 hours in media containing different concentrations of ab120449 (BAPTA sodium salt) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab66705 staining PAI1 in HepG2 cells treated with BAPTA-AM (ab120503), by ICC/IF. Increase in PAI1 expression correlates with increased concentration of BAPTA-AM, as described in literature.
The cells were incubated at 37°C for 4 hours in media containing different concentrations of ab120503 (BAPTA-AM) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab66705 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.