Anti-FOXC1 antibody [EPR20685]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] (AB227977)

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

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] (AB227977)

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

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] (AB227977)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FOXC1 antibody [EPR20685] (AB227977)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] (AB227977)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FOXC1 antibody [EPR20685] (AB227977)

ab227977 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 ab227977 at 12 μg/ml 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)

Lab

Flow Cytometry (Intracellular) - Anti-FOXC1 antibody [EPR20685] (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.

• IP

Unknown

Immunoprecipitation - Anti-FOXC1 antibody [EPR20685] (AB227977)

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.

All lanes:

Immunoprecipitation - Anti-FOXC1 antibody [EPR20685] (ab227977)

Predicted band size: 57 kDa

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• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-FOXC1 antibody [EPR20685] (AB227977)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXC1 antibody [EPR20685] (AB227977)

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

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

• WB

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Western blot - Anti-FOXC1 antibody [EPR20685] (AB227977)

Exposure time : Lanes 1 & 4 : 3 minutes; Lanes 2-3 : 5 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular mass is consistent with what has been described in the literature (PMID : 27708239).

All lanes:

Western blot - Anti-FOXC1 antibody [EPR20685] (ab227977) at 1/1000 dilution

Lane 1:

MDA-MB-231 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Human fetal kidney lysate at 10 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 1/50000 dilution

Predicted band size: 57 kDa

Observed band size: 70 kDa

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• WB

CiteAb

Western blot - Anti-FOXC1 antibody [EPR20685] (AB227977)

FOXC1 western blot using anti-FOXC1 antibody [EPR20685] ab227977. Publication image and figure legend from Bao, C., Lu, Y., et al., 2019, Cell Death Dis, PubMed 31649243.

ab227977 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab227977 please see the product overview.

MiR-9-3p directly targets FOXC1/FAM171A1, and miR-135b-3p directly targets RGMA.a, b The dual luciferase assays in MDA-MB-231 cells confirmed that miR-9-3p directly targets FOXC1/FAM171A1, and miR-135b-3p directly targets RGMA. c–h The mRNA and protein levels of FOXC1, FAM171A1 and RGMA were measured in MDA-MB-231 with transfection of specific miRNA mimics. i The expression levels of FOXC1, FAM171A1 and RGMA were compared among normal (n = 113), TNBC (n = 115), non-TNBC (n = 973) samples form TCGA database. j The mRNA expressions of FOXC1, FAM171A1 and RGMA were measured in HBL-100 (normal), TNBC (MDA-MB-231, BCap37, Hs 578 T, BT-549, HCC1937) and non-TNBC (MCF-7) cell lines. Bars indicate the mean ± SD of three independent replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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