Anti-FOXP3 antibody [236A/E7]

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [236A/E7] (AB20034)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling FOXP3 with ab20034 at a concentration of 2 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab20034 anti-FOXP3 antibody [236A/E7] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

Immunohistochemical analysis of paraffin-embedded human thymus tissue labeling FOXP3 with ab23004 at 1/500 dilution followed by a ready to use Goat Anti-Mouse IgG. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is a ready to use Goat Anti-Mouse IgG.

Perform heat mediated antigen retrieval using Tris/EDTA buffer (pH 9.0).

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

FoxP3+ cells mainly accumulate centrally.

The formalin-fixed, paraffin-embedded blocks were cut into approximately <2 μm thick slices and mounted on SuperFrost Plus microscope slides (Menzel Gläser, Braunschweig, Germany). After deparaffinization and rehydration, sections were immersed into Dako Target Retrieval solution (Dako North America Inc., Carpinteria, USA), pH 6, 1/10, incubated at 97°C–99°C at 750 Watt for 2x 15 minutes, and allowed to cool to room temperature for 20 minutes. Endogenous peroxidase activity was blocked by 10-minute incubation in 7.5% hydrogen-peroxide solution (Hydroxen Peroxide Solution, Sigma Aldrich Co., Munich, Germany). Immunohistochemical staining for FoxP3 (1/180 dilution; for 60 min) was performed according to standard procedure using MACH-3 mouse alkalic phosphatase polymer detection kit from Biocare Medical Systems (Concord, USA). The slides were incubated with monoclonal mouse antibody. Chromogen Red (Dako North Amerika Inc., Carpinteria, USA) was used as chromogen for FoxP3 staining, and lastly hematoxylin counterstaining was done (Vector Laboratories, Burlingam, USA).

This image was generated from the hybridoma version of the product.

Hermans C. et al PLoS One. 2014 Nov 3;9(11):e111757. doi: 10.1371/journal.pone.0111757. eCollection 2014. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [236A/E7] (AB20034)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

Immunohistochemical analysis of formalin fixed paraffin embedded) human tonsil labelling FOXP3 with ab20034 at a concentration of 2 µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab20034 anti-FOXP3 antibody [236A/E7] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

AbReview16484****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

ab20034 staining FOXP3 in human colon tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval and then blocking with 10% serum for 2 hours at 21°C. The primary antibody was diluted 1/50 and incubated with sample for 2 hours at 21°C. An Alexa Fluor^®488-conjugated rabbit polyclonal to mouse IgG was used undiluted as secondary antibody.

This image was generated from the hybridoma version of the product.

This image is a courtesy of Nicole Schechter

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling FOXP3 with ab23004 at 1/500 dilution followed by a ready to use Goat Anti-Mouse IgG. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is a ready to use Goat Anti-Mouse IgG.

Perform heat mediated antigen retrieval using Tris/EDTA buffer (pH 9.0).

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [236A/E7] (AB20034)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

Immunohistochemical analysis of human large and locally advanced breast cancers staining FOXP3 using ab20034. (a) Low level of FOXP3⁺, CTLA-4⁺ Treg infiltration (b) High level of FOXP3⁺ and CTLA-4⁺ Treg infiltration. (Itu : intratumoral Str : stromal)

This image was generated from the hybridoma version of the product.

This image is from PubMedId: 27777963. Kaewkangsadan V et al. (2016) Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [236A/E7] (AB20034)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND^® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• mIHC

Collaborator

Multiplex immunohistochemistry - Anti-FOXP3 antibody [236A/E7] (AB20034)

Fluorescence multiplex immunohistochemical analysis of Human breast cancer tissue (formalin-fixed paraffin-embedded section).

Merged staining of Anti-PD-L1 (ab251611; cyan; Opal™ 520), Anti-Granzyme B (ab219803; yellow; Opal™ 540), Anti-PD1 (ab251613; magenta; Opal™ 570), Anti-pan Cytokeratin (ab264485; red; Opal™ 620), Anti-EpCAM (ab225894; red; Opal™ 620), Anti-CD8 alpha (ab251596; green; Opal™ 650) and Anti-FOXP3 (ab96048; orange; Opal™ 690). EpCAM and pan-cytokeratin share the same dye and color. Dyes are pseudo-colored for better contrast of the markers.

The immunostaining was performed on a Leica Biosystems BOND® MAX instrument with an Opal™ 6-Plex Detection Kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; sequentially for ab251611 (1/750 dilution), ab219803 (1/250 dilution), ab251613 (1/750 dilution), ab264485 (0.5 μg/ml), ab225894 (1/1250 dilution), ab251596 (1/1500 dilution) and ab96048 (10 μg/ml); each using a separate fluorescent tyramide signal amplification system. EDTA based antigen retrieval (Leica Biosystems BOND^® Epitope Retrieval Solution 2, pH 9.0, 20 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity. DAPI (dark blue) was used as a nuclear counter stain.
Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra 3 Imaging System (Akoya Biosciences^®).

This data is courtesy of ImmunoAtlas and it can be found here.

This image is courtesy of ImmunoAtlas.

• WB

Lab

Western blot - Anti-FOXP3 antibody [236A/E7] (AB20034)

ab20034 detects Human FOXP3 protein at ~50 kDa in HEK293T cells overexpressing the protein. It also detects FOXP3 in Human tonsil tissue lysate, however this band is significantly weaker in endogenous conditions. Upon higher exposure, weak bands can also be observed in HEK293T cell lysate.

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-FOXP3 antibody [236A/E7] (ab20034; 5 microgram per mL) overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-mouse (green; 1 : 10000) for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FOXP3 antibody [236A/E7] (ab20034) at 5 µg/mL

Lane 1:

HEK293T cell lysate at 20 µg

Lane 2:

HEk293T cell lysate overexpressing Human FOXP3 at 20 µg

Lane 3:

Human tonsil tissue lysate at 20 µg

Predicted band size: 47 kDa

false

• WB

Unknown

Western blot - Anti-FOXP3 antibody [236A/E7] (AB20034)

All lanes:

Western blot - Anti-FOXP3 antibody [236A/E7] (ab20034) at 1/1000 dilution

All lanes:

Human mammary gland lysate at 20 µg

Predicted band size: 47 kDa

false

• IHC-P

AbReview30757****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP3 antibody [236A/E7] (AB20034)

ab20034 staining FOXP3 in Cynomolgus Monkey Spleen tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% donkey serum for 20 minutes at room temperature; antigen retrieval was by heat mediation using EDTA pH9.0. Samples were incubated with primary antibody (1/100 dilution) for 30 minutes. A Biotin-conjugated Donkey anti-mouse polyclonal (1/2000 dilution) was used as the secondary antibody.

This image was generated from the hybridoma version of the product.

Image is courtesy of an AbReview submitted by Jing Ma

• WB

Unknown

Western blot - Anti-FOXP3 antibody [236A/E7] (AB20034)

All lanes:

Western blot - Anti-FOXP3 antibody [236A/E7] (ab20034) at 1/1000 dilution

Lane 1:

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

Lane 2:

HEK-293 transfected with FOXP3 (WT) expression vector containing a GFP-Myc-tag, whole cell lysate at 10 µg

Predicted band size: 47 kDa

false

Exposure time: 1s