Immunohistochemical analysis of paraffin-embedded human small cell lung cancer tissue labeling DLL3 with ab229902 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human small cell lung cancer (PMID: 28487384, 30397180) is observed.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

The section was incubated with ab229902 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Anti-DLL3 antibody [EPR22592-18] (ab229902) at 1/1000 dilution + TT (human thyroid carcinoma epithelial cell) whole cell lysate at 10 µg with 5% NFDM/TBST

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 65 kDa
Observed band size: 65 kDa

Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling DLL3 with ab229902 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human glioma (PMID: 30397180) is observed.

The section was incubated with ab229902 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded mouse embryonic brain of day 14.5 tissue labeling DLL3 with ab229902 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse embryonic brain of day 14.5 (PMID: 19389376) is observed.

The section was incubated with ab229902 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling DLL3 with ab229902 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human cerebrum (PMID: 30397180) is observed.

The section was incubated with ab229902 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling DLL3 with ab229902 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on mouse cerebrum (PMID: 30397180) is observed.

The section was incubated with ab229902 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with DLL3 expression construct containing a myc-His-tag^®  labelling DLL3 with ab229902 at 1/600 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730, Left) isotype control. 

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab229902 (Right). Then stained with anti-myc-tag conjugated to Alexa Fluor^® 647.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells labelling DLL3 with ab229902 at 1/100 dilution, followed by ab150080 AlexaFluor^®594 Goat anti-Rabbit secondary antibody at 1/500 dilution (Green). An anti-myc-tag mAb (Alexa Fluor^® 488 Conjugate) was used at 1/100 dilution. Confocal image showing positive staining in HEK-293T cells transfected with DLL3 expression construct containing a myc-His-tag^® is observed.  The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150080 AlexaFluor^®594 Goat anti-Rabbit secondary at 1/500 dilution.

DLL3 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) transfected with DLL3 expression construct containing a myc-His-tag®  whole cell lysate with ab229902 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab229902 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HEK-293T (human embryonic kidney epithelial cell) transfected with DLL3 expression construct containing a myc-His-tag®  whole cell lysate 10μg

Lane 2: ab229902 IP in HEK-293T transfected with DLL3 expression construct containing a myc-His-tag^®  whole cell lysate (Input).

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab229902 in HEK-293T transfected with DLL3 expression construct containing a myc-His-tag®  whole cell lysate.

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

Exposure time: 15 seconds. 

All lanes : Anti-DLL3 antibody [EPR22592-18] (ab229902) at 1/1000 dilution

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DLL3 (WT) expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with empty vector containing myc-His-tag®, whole cell 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: 65 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.