ab23345 at 1 mg/ml + Human ES Cells treated with Retinoic Acid (48h) at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?
Additional bands at: 50 kDa, 65 kDa, 75 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab23345 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Abcam recommends using milk as the blocking agent.

Dlx2 and Tbr2 identify non overlapping progenitor lineages in the adult mouse SVZ (subventricular zone).

(A–B) Adult C57/BL6 mouse forebrain coronal sections at rostro-caudal point 1.2 relative to the bregma were immunostained for Dcx (red), Dlx2 or Tbr2 (green) and Ki67 (blue). Both progenitor populations show characteristics of migrating neuroblasts, as indicated by their Dcx expression (C–D). Adult Mash1 mouse forebrain coronal sections at rostro-caudal point 1.2 relative to the bregma were immunostained for Tbr2 (red) and Dlx2 (blue). Both Tbr2 and Dlx2 exhibited EGFP expression, but showed no colocalisation. Right side captions show cropped individual channels and the merges. Full panel insets are zoomed and cropped DAB stained photomicrographs of rostral periventricular sections for Tbr2 in the dorsal SVZ (A), Dlx2 in the dorso-lateral SVZ (B) and Mash1 in the ventro-lateral SVZ (C). Yellow arrows and arrowheads show respectively positive stained cell and low level TF staining. Dotted lines mark approximate boundaries of ventricular space. Flattened confocal z-stacks are of 14–15 µm thickness, including captions. Scale bars: 15 µm in full panels, 20 µm in captions and 25 µm in insets.

Anti-TBR2 / Eomes antibody (ab23345) at 1 µg/ml + Human ES Cells treated with Retinoic Acid (24h) at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?
Additional bands at: 75 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab23345 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Abcam recommends using milk as the blocking agent.

IHC image of TRB2 staining in a mouse brain E14 frozen tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6). Non-specific protein-protein interactions were then blocked in TBS containing 0.2% (v/v) Triton X-100 for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.05% (v/v) Triton X-100 and 1% (w/v) BSA with ab23345 at 1/100 dilution. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody was used to detect the primary antibody.  The section was mounted with DPX.

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.

All lanes : Anti-TBR2 / Eomes antibody (ab23345) at 1 µg/ml (BLOCKED WITH 3% MILK)

Lane 1 : Human Mesendoderm (Day 2) Whole Cell Lysate
Lane 2 : E14 Mouse Embryo Brain Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?
Additional bands at: 75 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

ab23345 staining mouse developing cerebral cortex tissue sections by IHC-Fr.  Sections were PFA fixed and permeabilized in TX-100 prior to blocking with 2.5% serum for 1 hour at RT.  The primary antibody was diluted 1/500 and incubated with the sample for 18 hours.  A biotinylated pig anti-rabbit IgG antibody, diluted 1/500, was used as the secondary.

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ab23345 staining TBR2 / Eomes in mouse embryonic brain tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with formaldehyde and blocking with 1% BSA and normal Goat serum for 30 minutes at RT. The sample was incubated with primary antibody (1/1000 in TBS + BSA 1%) for 10 hours at 40C. An Alexa Fluor® 555-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody at 1/800 dilution.

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Lanes 1-3 : Anti-TBR2 / Eomes antibody (ab23345) at 1/2000 dilution
Lanes 4-6 : V5 antibody

Lane 1 : EL4 cells + empty vector
Lane 2 : EL4 cells + vector expressing V5 tagged Eomesodermin
Lanes 3 & 6 : EL4 cells + V5 tagged vector
Lane 4 : EL4 cells + empty vector
Lane 5 : EL4 cells + vector expressing V5 tagged Eomesodermin

Secondary
All lanes : Goat anti Rabbit at 1/2500 dilution

Predicted band size: 72 kDa
Observed band size: 72 kDa

ab23345 detects a clear band of ~ 72 kDa in lysates from EL4 cells expressing V5 tagged Eomesodermin (lane 2).  Lanes 1 and 3 contain lysates from EL4 cells expressing empty vector or V5 tag alone. Lanes 4-6 show the same lysates blotted with anti-V5 tag antibody. GAPDH was used as a loading control.

All lanes : Anti-TBR2 / Eomes antibody (ab23345) at 1 µg/ml

Lane 1 : Human Mesendoderm (Day 2) Whole Cell Lysate
Lane 2 : Human Mesendoderm (Day 2) Whole Cell Lysate with Mouse TBR2 / Eomes peptide (ab25698) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?
Additional bands at: 74 kDa. We are unsure as to the identity of these extra bands.

Anti-TBR2 / Eomes antibody (ab23345) at 1/1000 dilution + Lysate prepared from mouse embryonic brain tissue at 20 µg

Secondary
HRP-conjugated goat polyclonal to rabbit IgG

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 72 kDa
Observed band size: 72 kDa


Exposure time: 5 minutes

See Abreview