重组Anti-Neuroligin 2抗体[RM2048] (ab317510)

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

Negative control: liver, kidney (PMID: 10718198, 8576240, 18434543).

Samples are non-boiled as boiling may cause protein aggregation.

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

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Neuroligin 2 with ab317510 at 1/2000 (0.249 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID: 19926856).
The section was incubated with ab317510 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 Neuroligin 2 with ab317510 at 1/2000 (0.249 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum (PMID: 16982420).
The section was incubated with ab317510 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 Neuroligin 2 with ab317510 at 1/2000 (0.249 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control: no staining on mouse liver.
The section was incubated with ab317510 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 Neuroligin 2 with ab317510 at 1/2000 (0.249 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control: no staining on rat liver.
The section was incubated with ab317510 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 (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a mouse NLGN2 expression vector containing a his tag. (B) HEK-293T cells transfected with a mouse NLGN1 expression vector containing a his tag. (C) HEK-293T cells transfected with a mouse NLGN3 expression vector containing a his tag. (D) HEK-293T cells transfected with empty vector containing a his tag. tissue labeling Neuroligin 2 with ab317510 at 1/2000 (0.249 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

This antibody does not cross-react with human NLGN1 and NLGN3.
Positive staining on (A) HEK-293T cells transfected with a mouse NLGN2 expression vector containing a his tag, no staining on (B) HEK-293T cells transfected with a mouse NLGN1 expression vector containing a his tag, and (C) HEK-293T cells transfected with a mouse NLGN3 expression vector containing a his tag, and (D) HEK-293T cells transfected with empty vector containing a his tag.
The section was incubated with ab317510 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 hippocampus (fresh frozen) tissue labeling Neuroligin 2 with ab317510 at 1/50 (9.94 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A: merged staining of anti-Neuroligin-2 (ab317510, green), anti-NeuN (ab190565, grey) and anti-GFAP (ab201732, magenta) on mouse hippocampus.
Panel B: anti-Neuroligin-2 stained on mouse hippocampus.
Panel C: anti-NeuN stained in neurons of mouse hippocampus.
Panel D: anti-GFAP stained in astrocytes of mouse hippocampus.
The section was incubated in two rounds of staining: in the order of ab317510 and ab190565, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). 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 Neuroligin 2 with ab317510 at 1/50 (9.94 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 ab317510 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 Rat hippocampus (fresh frozen) tissue labeling Neuroligin 2 with ab317510 at 1/50 (9.94 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A: merged staining of anti-Neuroligin-2 (ab317510, green), anti-NeuN (ab190565, grey) and anti-GFAP (ab201732, magenta) on rat hippocampus.
Panel B: anti-Neuroligin-2 stained on rat hippocampus.
Panel C: anti-NeuN stained in neurons of rat hippocampus.
Panel D: anti-GFAP stained in astrocytes of rat hippocampus.
The section was incubated in two rounds of staining: in the order of ab317510 and ab190565, ab201732 for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). 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 Rat liver (fresh frozen) tissue labeling Neuroligin 2 with ab317510 at 1/50 (9.94 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 rat liver. The nuclear counterstain was DAPI (Blue). The section was incubated with ab317510 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 neural/glia cell cells labelling Neuroligin 2 with ab317510 at 1/50 (9.94 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 positive staining in mouse primary neural/glia cell (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 um 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.

-ve control 1: ab317510 at 1/50 (9.94 ug/ml) dilution, followed by ab150120 at 1/1000 2 ug/mL dilution.
-ve control 2: ab11267 1/500 4ug/ml dilution, followed by ab150081 at 1/1000 2 ug/mL dilution (Green).

Flow cytometric analysis of Mouse primary neuron cells labelling Neuroligin 2 with ab317510 at 1/500 dilution (0.1ug)/right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

Flow cytometric analysis of Rat primary neuron cells labelling Neuroligin 2 with ab317510 at 1/500 dilution (0.1ug)/right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.

Neuroligin 2 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate with ab317510 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317510 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate
Lane 2: ab317510 IP in Mouse brain tissue lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317510 in mouse brain tissue lysate

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

Exposure time: 8 seconds.