Anti-JIP3 antibody [EPR30425-79]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-JIP3 antibody [EPR30425-79] (AB326560)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) and Differentiated SH-SY5Y cells labelling JIP3 with ab326560 at 1/100 (5.12 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 increased cytoplasmic staining in differentiated SH-SY5Y cells (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).

ab78078 Anti-beta III Tubulin mouse monoclonal antibody - Neuronal Marker was used to counterstain tubulin at 1/200 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling JIP3 with ab326560 at 1/500 (1.024 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling JIP3 with ab326560 at 1/500 (1.024 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive on human testis. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling JIP3 with ab326560 at 1/500 (1.024 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Low expression : Weak staining on human spleen. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling JIP3 with ab326560 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on mouse testis. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling JIP3 with ab326560 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on mouse cerebrum (PMID : 10629060). The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling JIP3 with ab326560 at 1/2000 (0.256 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : Very weak staining on mouse spleen (PMID : 10629060). The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-JIP3 antibody [EPR30425-79] (AB326560)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse primary neuron cells labelling JIP3 with ab326560 at 1/100 (5.12 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 mouse primary neuron(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).

Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab326560 at 1/100 dilution, followed by ab150120 at 1/1000 dilution.
-ve control 2 : ab11267 at 1/500 dilution, followed by ab150081 at 1/1000 dilution.

• WB

Lab

Western blot - Anti-JIP3 antibody [EPR30425-79] (AB326560)

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

This antibody does not cross-react with human MAPK8IP1, MAPK8IP2 or MAPK8IP4.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(240KDa).

All lanes:

Western blot - Anti-JIP3 antibody [EPR30425-79] (ab326560) at 1/1000 dilution

Lane 1:

293T cells transfected with a human MAPK8IP3 expression vector containing a his-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human MAPK8IP1 expression vector containing a his-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human MAPK8IP2 expression vector containing a his-tag, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human MAPK8IP4 expression vector containing a his-tag, whole cell lysate at 20 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

Observed band size: 240 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-JIP3 antibody [EPR30425-79] (AB326560)

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

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

All lanes:

Western blot - Anti-JIP3 antibody [EPR30425-79] (ab326560) at 1/1000 dilution

Lane 1:

Untreated SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

Neuronal differentiated SH-SY5Y for 14 days whole cell lysate at 50 µg

Lane 3:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 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

Observed band size: 240 kDa,36 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-JIP3 antibody [EPR30425-79] (AB326560)

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

Low expression : liver, spleen(PMID : 10629060)

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

Exposure time : Lanes 1-6 : 180 seconds, lanes 7-8 : 15 seconds

All lanes:

Western blot - Anti-JIP3 antibody [EPR30425-79] (ab326560) at 1/1000 dilution

Lane 1:

Mouse brain frozen tissue lysate at 20 µg

Lane 2:

Mouse cerebellum frozen tissue lysate at 20 µg

Lane 3:

Mouse testis tissue frozen lysate at 20 µg

Lane 4:

Mouse heart tissue frozen lysate at 20 µg

Lane 5:

Mouse liver frozen tissue lysate at 20 µg

Lane 6:

Mouse spleen frozen tissue lysate at 20 µg

Lane 7:

Mouse brain fresh tissue lysate at 20 µg

Lane 8:

Mouse liver fresh tissue lysate at 20 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

Observed band size: 240 kDa,36 kDa

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-JIP3 antibody [EPR30425-79] (AB326560)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocytes cells labelling JIP3 with ab326560 at 1/100 (5.12 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Low expression : Confocal image showing no staining in mouse splenocytes(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).Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

Anti-rat 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.