Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling Neurogranin with ab217672 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Mainly cytoplasmic staining on rat cerebrum (PMID: 26076492) is observed. Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-Neurogranin antibody [EPR21152] (ab217672) at 1/1000 dilution

Lane 1 : Human fetal brain lysate at 10 µg
Lane 2 : Human brain cortex lysate at 10 µg
Lane 3 : Mouse cerebral cortex lysate at 20 µg
Lane 4 : Rat brain lysate at 20 µg

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Lanes 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 8 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?

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

Exposure times:

Lane 1: 32 seconds

Lane 2: 101 seconds

Lane 3: 85 seconds

Lane 4: 3 minutes 

The observed molecular mass is consistent with the literature (PMID 17505539).

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse hippocampus CA1 tissue labeling Neurogranin with ab217672 at 1/3000 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1000 dilution. Positive staining in the stratum pyramidal neurons of hippocampus CA1 on mouse brain (PMID: 15389631; 21516261) is observed. Counter stained with DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1000 dilution.

Perform heat-mediated antigen retrieval by using sodium citrate buffer (10 mM citrate pH 6.0 + 0.05% Tween-20)

Immunocytochemistry/ Immunofluorescence analysis of primary rat cortical neurons labeling Neurogranin with ab217672 at 1/1500. The cells were fixed with 4% paraformaldehyde containing 0.2% picric acid in 0.1M phosphate buffer, pH 6.9 for 20 minutes. Permeabilization was with 0.3% Triton-X 100 in PBS (PBSTx). ab150068 at 1/200 was used as the secondary antibody

The rat cortical neurons were cultured for 29 days in vitro. They were either left untreated (Control) or treated beginning on the 10^th day with 60ng/mL triiodothyronine (T3), to enhance neurogranin expression.

The cells were visualized with an inverted microscope at 10X magnification (upper panels) or 20X magnification (lower panels).

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized  frozen rat hippocampus CA1 tissue labeling Neurogranin with ab217672 at 1/3000 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1000 dilution. Positive staining in the stratum pyramidal neurons of hippocampus CA1 on rat brain (PMID: 15389631; 21516261) is observed. Counter stained with DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1000 dilution.

Perform heat-mediated antigen retrieval by using sodium citrate buffer (10 mM citrate pH 6.0 + 0.05% Tween-20)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling Neurogranin with ab217672 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Mainly cytoplasmic staining on mouse cerebrum (PMID: 26076492) is observed. Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Neurogranin with ab217672 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Mainly cytoplasmic but also weak nuclear staining on human cerebrum (PMID: 26076492; PMID: 21516261) is observed. Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Neurogranin was immunoprecipitated from 0.35 mg mouse cerebral cortex lysate with ab217672 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217672 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution.

Lane 1: Mouse cerebral cortex lysate 10 µg (Input).
Lane 2: ab217672 IP in mouse cerebral cortex lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab217672 in mouse cerebral cortex lysate (-).

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 15 seconds.