IHC image of FoxG1 staining in mouse brain E14 formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. 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 ab18259, 0.5 µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

Staining revealed in telencephalon (but not diencephalon) as expected.

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-FOXG1 antibody (ab18259) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : E10 Mouse Embryo Brain Tissue Lysate
Lane 4 : Brain (Rat) Tissue Lysate
Lane 5 : Human spleen tissue lysate - total protein (negative control)

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 50 kDa
Additional bands at: 155 kDa, 52 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 3 minutes

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 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 ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes : Anti-FOXG1 antibody (ab18259) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : Brain (Rat) Tissue Lysate
Lane 4 : Human brain tissue lysate - total protein (ab29466) with Human FOXG1 peptide (ab19644) at 1 µg/ml
Lane 5 : Brain (Mouse) Tissue Lysate with Human FOXG1 peptide (ab19644) at 1 µg/ml
Lane 6 : Brain (Rat) Tissue Lysate with Human FOXG1 peptide (ab19644) at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 50 kDa


Exposure time: 1 minute

ab18259 detects a band of ~50kDa in brain lysates. This band is blocked by addition of the immunizing peptide ab19644 which suggests that is specific for FOXG1.

 

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 5% Bovine Serum Albumin before being incubated with ab18259 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution.

ab18259 staining Foxg1 (1/50) in the telencephalon (but not the diencephalon) as expected. DAB-immunohistochemistry was performed on embryonic (E13.5) mouse brain (coronal paraffin-embedded sections) after microwave treatment with 10mM sodium citrate.

ab18259 staining FOXG1 in human A549 cells by immunocytochemistry/immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized using 0.25% Triton, blocked with 1% BSA for 1 hour at room temperature and then incubated with ab18259 at a 1/400 dilution for 1 hour. The secondary used was an Alexa-Fluor 488 conjugated goat polyclonal used at a 1/250 dilution.

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