Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human striatum* performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen.

The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

Negative control image : IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human liver* performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

IHC image of Dopamine Transporter staining in a section of formalin fixed paraffin embedded normal human substantia nigra* performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was incubated with ab184451, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Dopamine Transporter with ab184451 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Counter stained with Hematoxylin.

Negative control : Negative staining on mouse liver.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of paraffin-embedded Rat striatum tissue labeling Dopamine Transporter with ab184451 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on rat striatum is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen Rat brain (sagittal section) tissue labeling Dopamine Transporter with ab184451 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The result showed cytoplasmic staining on rat striatum.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling Dopamine Transporter with ab184451 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Counter stained with Hematoxylin.

Negative control : Negative staining on rat spleen.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen Mouse brain (Coronal section) tissue labeling Dopamine Transporter with ab184451 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The result showed cytoplasmic staining on mouse striatum but negative on lateral ventricle (LA).

The nuclear counterstain is DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Immunohistochemical analysis of paraffin-embedded Mouse striatum tissue labeling Dopamine Transporter with ab184451 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membrane and cytoplasm staining on mouse striatum is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

• IP

Supplier Data

Immunoprecipitation - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Dopamine Transporter was immunoprecipitated from 0.35mg of Mouse striatum whole cell lysate with ab184451 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab184451 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Mouse striatum whole cell lysate, 10μg (Input).

Lane 2 : ab184451 IP in Mouse striatum whole cell lysate.

Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab184451 in Mouse striatum whole cell lysate.

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

Exposure time : 30 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

All lanes:

Immunoprecipitation - Anti-Dopamine Transporter antibody [EPR19695] (<a href='/en-us/products/primary-antibodies/dopamine-transporter-antibody-epr19695-ab184451'>ab184451</a>)

Predicted band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

Blocking/Dilution buffer : 5% NFDM/TBST.

ab181602 was used as GAPDH loading control.

Dopamine transporters are distributed widely across the brain in regions with high dopaminergic activity, such as the striatum and substantia nigra (PMID : 1765147), so non-related tissues and cells were chosen as negative controls; and DAT is a glycoprotein with molecular weight of 70-85KD (PMID : 25356398, PMID : 20643191).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

All lanes:

Western blot - Anti-Dopamine Transporter antibody [EPR19695] (<a href='/en-us/products/primary-antibodies/dopamine-transporter-antibody-epr19695-ab184451'>ab184451</a>) at 1/1000 dilution

Lane 1:

Mouse striatum lysate at 20 µg

Lane 2:

Rat striatum lysate at 20 µg

Lane 3:

Mouse kidney lysate at 20 µg

Lane 4:

Rat liver lysate at 20 µg

Lane 5:

Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate at 20 µg

Lane 6:

C6 (Rat glial tumor cell line) 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

Predicted band size: 68 kDa

Observed band size: 70-85 kDa

false

Exposure time: 3min

• WB

Unknown

Western blot - Anti-Dopamine Transporter antibody [EPR19695] - BSA and Azide free (AB221845)

We recommend not to boil the samples after lysis to get desired WB results.

ab181602 was used as a GAPDH loading control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab184451).

All lanes:

Western blot - Anti-Dopamine Transporter antibody [EPR19695] (<a href='/en-us/products/primary-antibodies/dopamine-transporter-antibody-epr19695-ab184451'>ab184451</a>) at 1/1000 dilution

Lane 1:

Mouse striatum lysate boiled at 20 µg

Lane 2:

Mouse striatum lysate unboiled at 20 µg

Lane 3:

Rat striatum lysate boiled at 20 µg

Lane 4:

Rat striatum lysate unboiled 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 1/20000 dilution

Predicted band size: 68 kDa

false