Anti-FOXC1 antibody [EPR20685] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells labeling FOXC1 with ab227977 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HEK-293T cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunohistochemical analysis of paraffin-embedded human basal-like breast cancer tissue labeling FOXC1 with ab227977 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in human basal-like breast cancer (PMID : 27708239; PMID : 20406990). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

Flow cytometry overlay histogram showing left HEK293 positive cells and right negative Jurkat stained with ab227977 (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 (ab227977) (1x 106 in 100μl at 1.0μg/ml (1/2110)) 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 HEK293 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

ab230159 staining FOXC1 in wild-type HEK293 cells (top panel) and FOXC1 knockout HEK293 cells (bottom panel). The cells were fixed with paraformaldehyde (10 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 ab230159 at 12 μg/ml concentration and ab7291 (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 pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized �HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line labeling �FOXC1 with ab227977 at 1/100 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^� 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling FOXC1 with ab227977 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HEK-293T cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling FOXC1 with ab227977 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in human gastric cancer (PMID : 24329718). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Unknown

Immunoprecipitation - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

FOXC1 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab227977 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab227977 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).

Lane 2 : ab227977 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab227977 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

All lanes:

Immunoprecipitation - Anti-FOXC1 antibody [EPR20685] (<a href='/en-us/products/primary-antibodies/foxc1-antibody-epr20685-ab227977'>ab227977</a>)

Predicted band size: 57 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling FOXC1 with ab227977 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in the pericytes and endothelium of blood vessels in mouse cerebrum is observed (PMID : 25733312; PMID : 23862012). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

Heat mediated antigen retrieval was performed with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse fetal brain E14.5 tissue labeling FOXC1 with ab227977  at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive nuclear staining localized in the meninges and adjacent cortex region on mouse fetal brain (PMID : 23862012).

The nuclear counter stain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227977).

• WB

Lab

Western blot - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

This data was developed using ab227977, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FOXC1 antibody [EPR20685] (<a href='/en-us/products/primary-antibodies/foxc1-antibody-epr20685-ab227977'>ab227977</a>) at 1/1000 dilution

Lane 1:

Mouse embryo tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse skin tissue lysate at 20 µg

Lane 4:

C2C12 (mouse myoblasts myoblast), whole cell lysate at 20 µg

Lane 5:

4T1 (mouse mammary gland carcinoma epithelial cell line) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 70 kDa

false

• WB

Lab

Western blot - Anti-FOXC1 antibody [EPR20685] - BSA and Azide free (AB230159)

This data was developed using ab227977, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FOXC1 antibody [EPR20685] (<a href='/en-us/products/primary-antibodies/foxc1-antibody-epr20685-ab227977'>ab227977</a>) at 1/1000 dilution

Lane 1:

Rat E15 brain tissue lysate at 20 µg

Lane 2:

Rat embryo tissue lysate at 20 µg

Lane 3:

Rat E18 kidney tissue lysate at 20 µg

Lane 4:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 70 kDa

false