Recombinant Anti-VGLUT2 antibody [EPR21085] (ab216463)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse midbrain neuron cells labelling VGLUT2 with ab216463 at 1/250 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing positive staining in mouse midbrain neuron cell. 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 (4 µg/mL) followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (2 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling VGLUT2 with ab216463 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Granularly cytoplasmic staining in mouse cerebrum is observed (PMID: 24804702). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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 sections of mouse thalamus tissue labeling VGLUT2 with ab216463  at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive staining in mouse thalamus.

The nuclear counter stain is DAPI (blue). 

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized rat midbrain neuron cells labelling VGLUT2 with ab216463 at 1/250 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing positive staining in rat midbrain neuron cell. 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 (4 µg/mL) followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (2 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).

Blocking/Dilution buffer: 5% NFDM/TBST.

Target is expressed in neurons, with spleen acting as a negative control (PMID: 15682395, PMID: 15102489). Note that the lysates were not boiled prior to gel loading to avoid aggregation of this membrane protein. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.

VGLUT2 was immunoprecipitated from 0.35 mg of mouse brain lysate with ab216463 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216463 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

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

Lane 2: ab216463 IP in mouse brain lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab216463 in mouse brain lysate.

Exposure time: 10 seconds.

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

Immunohistochemical analysis of paraffin-embedded mouse brain tissue labeling VGLUT2 with ab216463 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse forebrain, including cerebral cortex and diencephalon is observed (PMID: 16217795). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

Immunohistochemical analysis of paraffin-embedded rat brain tissue labeling VGLUT2 with ab216463 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on rat forebrain including cerebral cortex and diencephalon is observed (PMID: 16217795). Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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 sections of rat cortex tissue labeling VGLUT2 with ab216463  at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive staining in rat cortex.

The nuclear counter stain is DAPI (blue). 

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Note that the lysates were not boiled prior to gel loading to avoid aggregation of this membrane protein. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.