IHC image of Doublecortin staining in a formalin fixed, paraffin embedded normal rat hippocampus (SVZ region) tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab207175 at 1/500 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. As a negative control (inset), an identical assay was performed without adding the primary antibody.

 

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunohistochemical analysis of paraffin-embedded mouse adult hippocampus tissue labeling Doublecortin with ab207175 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on mouse hippocampal dentate gyrus is observed [PMID:23690918] [PMID:16814555]. 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 adult hippocampus tissue labeling Doublecortin with ab207175 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on rat hippocampal dentate gyrus is observed [PMID:23690918] [PMID:16814555]. 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 rat adult hippocampus tissue labeling Doublecortin with ab207175 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining on rat hippocampal dentate gyrus is observed [PMID:23690918] [PMID:16814555].  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) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 100% Methanol-fixed SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells labeling Doublecortin with ab207175 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on SH-SY5Y cell line.

The nuclear counter stain 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-Doublecortin antibody [EPR19997] (ab207175) at 1/10000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human brain 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: 40 kDa


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The higher molecular weight band is phosphorylated form and the lower molecular weight band is non-phosphorylated form. (PMID: 17178868).

Doublecortin is highly expressed during embryonic development and downregulated in adult tissues. (PMID: 10399933).

All lanes : Anti-Doublecortin antibody [EPR19997] (ab207175) at 1/10000 dilution

Lane 1 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
Lane 2 : Mouse brain 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: 40 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 15 seconds; Lane 2: 3 minutes.

The higher molecular weight band is phosphorylated form and the lower molecular weight band is non-phosphorylated form. (PMID: 17178868).

Doublecortin is highly expressed during embryonic development and downregulated in adult tissues. (PMID: 10399933).

All lanes : Anti-Doublecortin antibody [EPR19997] (ab207175) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Mouse brain lysate
Lane 4 : Mouse heart lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat heart 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: 40 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1-4: 3 minutes; Lane 5/6: 10 seconds.

The observed expression profile is consistent with what has been described in the literature (PMID: 10550327).