This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of (A) His-human TrkA overexpression 293T whole cell pellet, (B) His-human TrkB overexpression 293T whole cell pellet, (C) His-human TrkC overexpression 293T whole cell pellet and (D) HEK-293T transfected with empty plasmid labelling Pan Trk with purified ab76291 at 1/1000. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. 

Positive staining on (A) His-human TrkA overexpression 293T whole cell pellet, (B) His-human TrkB overexpression 293T whole cell pellet and (C) His-human TrkC overexpression 293T whole cell pellet.
No staining on (D) HEK-293T transfected with empty plasmid.
The section was incubated with abab76291 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

All lanes : Anti-Pan Trk antibody [EP1058Y] (ab76291) at 1/1000 dilution

Lanes 1 & 3 & 5 : Empty vector over expression 293T whole cell lysates
Lane 2 : His-human TrkA overexpression 293T whole cell lysates
Lane 4 : His-human TrkB overexpression 293T whole cell lysates
Lane 6 : His-human TrkC overexpression 293T whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 87 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

This antibody has relatively lower affinity to TrkC compared to TrkA and TrkB.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.  Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on human cerebrum. 
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunocytochemsitry/Immunofluorescence analysis of 293T (human embryonic kidney epithelial cell) cells labelling Pan Trk with ab76291 at 1/1000 dilution (0.7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkA expression vector.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Overlay histogram showing SH-SY5Y cells stained with unpurified ab76291 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76291, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with methanol (5 min) used under the same conditions.
Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunocytochemistry/immunofluorescence analysis of SH-SY5Y (Human neuroblastoma epithelial cell) labeling pan Trk with ab76291 at 1/100 dilution (7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/mL).

Confocal image showing cytoplasmic staining in SH-SY5Y cell line.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neuron cells labelling Pan Trk with ab76291 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

ab76291 at 1/40 immunoprecipitating Pan Trk in mouse brain tissue lysate observed at 145 kDa.

Lane 1 (input): Mouse brain tissue lysate (10µg)
Lane 2 (+): ab76291+ mouse brain tissue lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76291 in Mouse brain lysate
For western blotting, ab76291 at 1/1000 dilution (0.7 μg/mL) and VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 were used.

The 30 kDa band is an intracellular fragment, and the 140 kDa observed MW which is higher than the predicted one is due to the glycosylation modification. (refer to ab189903).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunocytochemsitry/Immunofluorescence analysis of 293T (human embryonic kidney epithelial cell) cells labelling Pan Trk with ab76291 at 1/500 dilution (1.4 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkC expression vector.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunocytochemsitry/Immunofluorescence analysis of 293T (human embryonic kidney epithelial cell) cells labelling Pan Trk with ab76291 at 1/1000 dilution (0.7 μg/mL). ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI (blue) was used as nuclear counterstain. Cells were counterstained with Myc-Tag (9B11) Mouse mAb (Alexa Fluor^® 647 Conjugate) at 1/200 (2.5 μg/mL). 

Confocal image showing cytoplasmic staining in 293T cells transfected with a myc-tagged hTrkB expression vector.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

This data was developed using ab76291, the same antibody clone in a different buffer formulation.

ELISA analysis of Human TrkA recombinant protein at 250 ng/mL with ab76291. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Negative control: No staining on human liver.

The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on rat cerebrum. 
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue labelling Pan Trk with purified ab76291 at 1/1000. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Heat mediated antigen retrieval was performed using Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes. Sections were counterstained with hematoxylin. Negative control using PBS instead of primary antibody.

Positive staining on mouse cerebrum. 
The section was incubated with ab76291 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Immunocytochemistry/Immunofluorescence analysis of U87-MG cells labelling Pan Trk with purified ab76291 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody, ab150113, an Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/500).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

ICC/IF image of Pan Trk staining on culture of mouse DRG neurons using unpurified ab76291 (1/100). The cells were fixed using formaldehyde and permeabilized using 0.2% Triton X-100. The cells were blocked using 10% Goat serum for 1 hour at 22°C. Unpurified ab76291 was diluted 1/100 using PBS and incubated with the cells for 30 mins at 22°C. The secondary antibody used was Goat polyclonal to Rabbit IgG conjugated to Alexa Fluor^® 488 (1/1000). Neuron was stained using Beta III tubulin antibody (Alexa Fluor^® 647)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).

Unpurified ab76291 staining Pan Trk in murine brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
Tissue was fixed with formaldehyde, permeabilized with 0.1% Saponin/PBS and blocked with 4% serum for 30 minutes at 25°C, antigen retrieval was by heat mediation with a citrate buffer. Samples were incubated with primary antibody (1/150 in blocking buffer) for 16 hours at 4°C. A FITC-conjugated goat anti-rabbit polyclonal IgG (1/100) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76291).