Immunohistochemical analysis of paraffin-embedded human cerebral cortex tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weakly cytoplasmic staining on neurons of human cerebral cortex is observed [PMID: 22007133].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic and nuclear staining on SH-SY5Y cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebral cortex tissue labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasmic and nuclear staining on neurons of mouse cerebral cortex is observed.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-HuD + HuC antibody [EPR19098] (ab184267) at 1/2000 dilution

Lane 1 : His-tagged mouse HuC recombinant protein (aa1-367)
Lane 2 : His-GST-tagged mouse HuD recombinant protein (aa1-385)
Lane 3 : His-tagged mouse HuR recombinant protein (aa1-360)
Lane 4 : His-tagged mouse HuB recombinant protein (aa1-360)

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa
Observed band size: 39,68 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 0.5 second.

Please note that the band around 55kDa in lane 2 is most likely the HuD protein with tags.

Flow cytometric analysis of 4% paraformaldehyde-fixed SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling HuC + HuD with ab184267 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

HuC + HuD was immunoprecipitated from 0.35 mg of mouse brain lysate with ab184267 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab184267 at 1/1000 dilution.

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

Lane 1: Mouse brain lysate, 10 µg (Input).

Lane 2: ab184267 IP in mouse brain lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab184267 in mouse brain lysate.

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

Exposure time: 30 seconds.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cell line) cells labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic and nuclear staining on Neuro-2a cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

All lanes : Anti-HuD + HuC antibody [EPR19098] (ab184267) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 40 kDa
Observed band size: 39 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile is consistent with what has been described in the literature (PMID: 9016658; PMID:8535975).

All lanes : Anti-HuD + HuC antibody [EPR19098] (ab184267) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Rat brain lysate
Lane 5 : Rat heart lysate
Lane 6 : Rat kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa
Observed band size: 39 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3: 1 minute; Lane 4-6: 5 seconds.

The expression profile is consistent with what has been described in the literature (PMID: 9016658; PMID: 8535975).

All lanes : Anti-HuD + HuC antibody [EPR19098] (ab184267) at 1/5000 dilution

Lane 1 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 2 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa
Observed band size: 39 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution Buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weakly cytoplasmic staining on myenteric ganglia of human colon is observed [PMID: 16918730].

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weakly cytoplasmic staining on human glioma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weakly cytoplasmic staining on neurons of mouse cerebral cortex is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and nuclear staining on myenteric ganglia of mouse stomach is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded rat cerebral cortex tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear and weakly cytoplasmic staining on neurons of rat cerebral cortex is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling HuC + HuD with ab184267 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and nuclear staining on myenteric ganglia of rat colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse stomach tissue labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasmic and nuclear staining on myenteric ganglia of mouse stomach is observed (white arrow).

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebral cortex tissue labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasmic and nuclear staining on neurons of rat cerebral cortex is observed.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen rat colon tissue labeling HuC + HuD with ab184267 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Cytoplasmic and nuclear staining on myenteric ganglia of rat colon is observed.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed Neuro-2a (Mouse neuroblastoma cell line) cells labeling HuC + HuD with ab184267 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.