Anti-Brn-2 抗体 [EPR29141-41] (ab317750)

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

Negative control: HeLa, JEG-3 (PMID: 2739723).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 2739723).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

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

Negative control: heart, liver (PMID: 2739723).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 2739723).

The identity of the higher MW band at approximately 75 kDa (in lane 2) and the lower MW band at approximately 40 kDa (in lane 3) are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Brn-2 with ab317750 at 1/100 (4.91 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining in human cerebrum.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Embryonic 20.5 mouse brain tissue labeling Brn-2 with ab317750 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining in embryonic 20.5 mouse brain.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Brn-2 with ab317750 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining in mouse cerebrum.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Brn-2 with ab317750 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Positive staining in rat cerebrum.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Brn-2 with ab317750 at 1/100 (4.91 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Negative control: No staining in human liver.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Brn-2 with ab317750 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Negative control: No staining in mouse liver.
The section was incubated with ab317750 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

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Brn-2 with ab317750 at 1/1000 (0.491 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond ™ Polymer Refine Detection).

Negative control: No staining in rat liver.
The section was incubated with ab317750 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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse E14.5 brain (fresh frozen) tissue labeling Brn-2 with ab317750 at 1/50 (9.82 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining on mouse E14.5 brain. The nuclear counterstain was DAPI (Blue). The section was incubated with ab317750 for 60 mins at room temperature. The section was then mounted using Fluoromount ®.The immunostaining was performed on a Leica Biosystems BOND ® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling Brn-2 with ab317750 at 1/50 (9.82 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Negative control: confocal image showing no staining on mouse liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab317750 for 60 mins at room temperature. The section was then mounted using Fluoromount ®.The immunostaining was performed on a Leica Biosystems BOND ® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Brn-2 with ab317750 at 1/50 (9.82 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing nuclear staining in mouse primary neuron (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain tubulin at 1/500 4ug/ml 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 2 ug/ml dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocyte cells labelling Brn-2 with ab317750 at 1/50 (9.82 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Negative control: spleen (PMID:2739723)
Confocal image showing no staining in mouse splenocytes. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor ® 594) was used to counterstain tubulin at 1/200 2.5ug/ml 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 2 ug/ml dilution.

Brn-2 was immunoprecipitated from 0.35 mg A375 (human malignant melanoma epithelial cell) whole cell lysate with ab317750 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317750 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: A375 (human malignant melanoma epithelial cell) whole cell lysate
Lane 2: ab317750 IP in A375 (human malignant melanoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317750 in A375 whole cell lysate

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

Exposure time: 67 seconds.