Anti-FOXP3 antibody [221D] - BSA and Azide free

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

This data was developed using ab253297, the same antibody clone in a different buffer formulation.

The BOND™ Polymer Refine Detection System on the BOND RX Automated Stainer (DS9800, Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical labelling of formalin-fixed, paraffin-embedded (FFPE) tissues. The protocol includes a peroxide block, post-primary reagent, polymer detection, DAB chromogen, and haematoxylin counterstain, all automated to minimize variability. Antigen retrieval was performed by heat-induced epitope retrieval (HIER) for 20 minutes in Tris-EDTA buffer (BOND Epitope Retrieval Solution 2, AR9640, Leica Biosystems, Wetzlar, Germany). For mouse and rabbit antibodies, the sequence comprised peroxide block (10 min), primary antibody incubation (30 min), post-primary (10 min), polymer (10 min), DAB (10 min), and haematoxylin (8 min). For rat antibodies, an anti-rat step (1 : 300 dilution, Vector Laboratories, California, USA; 20 min) was included prior to polymer application (15 min).

For mouse tissues, antigen retrieval was performed using high pH buffers (Dako, Agilent), followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide. Slides were then incubated with the primary antibody. Antigen/antibody complexes detection was carried out using a horseradish peroxidase-conjugated visualization system (Novolink Polymer, Leica). Staining was developed with 3,3′-diaminobenzidine (EnVision FLEX DAB Enhancer, Dako, Agilent), and nuclei were counterstained with hematoxylin (FLEX Hematoxylin, SM806, Dako, Agilent). Images were acquired using an Axiocam CCD camera (Zeiss) and processed with ZEN 2.1 software and Photoshop 9.0.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

Immunohistochemical analysis of paraffin-embedded mouse thymus tissue labeling FOXP3 with ab253297 at 1/1000 dilution (1.514 μg/ml) followed by a ready to use secondary. Nuclear staining on mouse thymus tissue. The section was incubated with ab253297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling FOXP3 with ab253297 at 1/1000 dilution (1.514 μg/ml) followed by a ready to use secondary. Nuclear staining on the stroma cells in the human colon. The section was incubated with ab253297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling FOXP3 with ab253297 at 1/1000 dilution (1.514 μg/ml) followed by a ready to use secondary. Nuclear staining on mouse spleen tissue. The section was incubated with ab253297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling FOXP3 with ab253297 at 1/1000 dilution (1.514 μg/ml) followed by a ready to use secondary. Nuclear staining on the human tonsil. The section was incubated with ab253297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

Immunohistochemical analysis of paraffin-embedded human stomach carcinoma tissue labeling FOXP3 with ab253297 at 1/1000 dilution (1.514 μg/ml) followed by a ready to use secondary. Nuclear staining on the stroma cells in the human stomach carcinoma. The section was incubated with ab253297 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

• WB

Lab

Western blot - Anti-FOXP3 antibody [221D] - BSA and Azide free (AB255942)

The molecular weight observed was in consistant with the literature (PMID : 17586580).

Blocking/Dilution buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab253297).

All lanes:

Western blot - Anti-FOXP3 antibody [221D] (<a href='/en-us/products/primary-antibodies/foxp3-antibody-221d-ab253297'>ab253297</a>) at 1.514 µg/mL

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a GFP-myc tag, whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with FOXP3 (WT) expression vector containing a GFP-myc tag, whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 47 kDa

Observed band size: 73 kDa

false

Exposure time: 3s