Anti-NFAT1 antibody [EPR30445-509]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) Human PBMC treated with Phorbol-12-myristate-13-acetate (20ng/ml) for 6hours and Ionomycin(0.5uM) for 6hours cells labelling NFAT1 with ab325917 at 1/50 (10.36 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in human PBMCs and nuclear staining in human PBMCs treated with Phorbol-12-myristate-13-acetate (20ng/ml) for 6hours and Ionomycin(0.5uM) for 6hours(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-human CD3 mouse monoclonal antibody (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on immune cells in human colon. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Daudi (human Burkitt's lymphoma lymphoblast) HeLa (human cervical adenocarcinoma epithelial cell) cells labelling NFAT1 with ab325917 at 1/50 (10.36 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in Daudi cell line and no staining in HeLa cell line(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Low expression : HeLa. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325917 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded Human Hodgkin Lymphoma tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human Hodgkin Lymphoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cervical carcinoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : very weak staining on human cerebrum. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

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

Low expression : HeLa, 293T, MCF7.

The identity of the bands lower than 75 kDa are unknown.

Lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross reactivity with human IgG at 1/2000 and lanes 3-8 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 48 seconds; Lanes 3-4 : 59 seconds; Lanes 5-8 : 180 seconds.

All lanes:

Western blot - Anti-NFAT1 antibody [EPR30445-509] (ab325917) at 1/1000 dilution

Lane 1:

Human lymph node tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 4:

Ramos (human burkitt's lymphoma b lymphocyte) whole cell lysate at 20 µg

Lane 5:

Daudi (human burkitt's lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 6:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 8:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Lanes 3 - 8:

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

Observed band size: 100-140 kDa,36 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NFAT1 antibody [EPR30445-509] (AB325917)

Immunohistochemical analysis of paraffin-embedded (A) : HEK-293T (human embryonic kidney epithelial cell) transfected with a human NFATC2 expression vector containing a myc-His-tag. (B) : HEK-293T transfected with empty vector containing a Myc-His tag tissue labeling NFAT1 with ab325917 at 1/2000 (0.259 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on (A) : HEK-293T transfected with a human NFATC2 expression vector containing a myc-His-tag, no staining on (B) : HEK-293T transfected with empty vector containing a Myc-His tag. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins