Anti-FOXP3 antibody [EPR22102-37]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methanol-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line transfected with a GFP-tagged FOXP3 expression construct labeling FOXP3 with ab215206 at 1/40 dilution (Right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 647) (ab150079), at 1/2000 dilution was used as the secondary antibody.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

10-color fluorescence multiplex immunohistochemical analysis of human lung cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-FOXP3 (ab215206; Cyan; TG540N), anti-PD1 (ab52587; Red; TG700N), anti-CD163 (ab182422; Brown; TG650N), anti-HLA-DR (ab92511; Yellow; TG570N), anti-CD4 (ab133616; Violet; TG620N), anti-CD8 alpha (ab101500; Purple; TG540S), anti-CD20 (ab9475; Grey; TG660S), anti-CD68 (ab192847; Green; TG520N), anti-Cytokeratin 19 (ab52625; Light blue; TG440N). TG470SN (dark blue) was used as a nuclear counter stain. The inset image shows the separate CD68 signal.

The section was incubated in nine rounds of staining; in the order of ab215206 (1/100 dilution), ab52587 (1/200 dilution), ab182422 (1/300 dilution), ab92511 (1/200 dilution), ab133616 (1/600 dilution), ab101500 (1/300 dilution), ab9475 (1/100 dilution), ab192847 (1/300 dilution), ab52625 (1/400 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (pH6.0) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

Image acquisition was performed with TissueFAXS Spectra (TissueGnostics).

This image is courtesy of TissueGnostics Asia Pacific Limited

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Human PBMCs were stained with Alexa Fluor^® 647 conjugated anti-human CD4 and BV421 conjugated anti-human CD25. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilization buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab98462, Dylight^®488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

Gating strategy and expression pattern is consistent with literature (PMID : 27330808).

• IP

Unknown

Immunoprecipitation - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

FOXP3 was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate with ab215206 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215206 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate 10 μg (Input).
Lane 2 : ab215206 IP in HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab215206 in HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate (-).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.

FOXP3 isoforms have been described in the literature. (PMID : 19568423).

All lanes:

Immunoprecipitation - Anti-FOXP3 antibody [EPR22102-37] (ab215206)

Predicted band size: 47 kDa,90 kDa

Observed band size: 55-73 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Rat splenocytes were stained with Alexa Fluor^® 647 conjugated anti-rat CD4. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilization buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab150077, Alexa Fluor^®488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Mouse splenocytes were stained with Alexa Fluor^® 647 conjugated anti-mouse CD4. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilisation buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

• WB

Unknown

Western blot - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a GFP-myc tag, whole cell lysate at 10 µg

Lane 2:

HEK-293T transfected with FOXP3 (WT) expression vector containing a GFP-myc tag, whole cell 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 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 73 kDa

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Exposure time: 6s

• WB

Unknown

Western blot - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Blocking buffer and concentration : 5% NFDM /TBST

Lane 1:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/200 dilution

Lane 2:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/1000 dilution

All lanes:

Full-length C-MYC/DDK tagged Recombinant Mouse Foxp3 protein 10ng

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

Predicted band size: 47 kDa

Observed band size: 50 kDa

false

Exposure time: 180s