Recombinant Anti-SOX10 antibody [SP267] (ab227680)

Flow cytometry overlay histogram showing left A-375 positive cells and right negative HeLa stained with ab227680 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab227680) (1x 10⁶ in 100μl at 0.2μg/ml (1/10250)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in A-375 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

ab227680 staining SOX10 in A375 cells, with negative expression in HeLa cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab227680 at 1 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 µg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

This product also work with 100% methanol (5 min) fixation under the same testing conditions.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human melanoma tissue sections labeling SOX10 with ab227680 at 1/100 dilution (0.25 μg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Mainly nuclear staining on the human melanoma, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab227680 for 10 mins at room temperature.

Immunohistochemistry (Frozen) analysis of mouse cerebellum tissue sections labeling SOX10 with purified ab227680 at 1/50 (1.4 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. Sections were fixed with 4% paraformaldehyde and permeabilised with 0.2% Triton X-100. Negative control: PBS instead of the primary antibody. DAPI (blue) was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat breast tissue sections labeling SOX10 with ab227680 at 1/100 dilution (0.25 μg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on the rat breast, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab227680 for 10 mins at room temperature.

Intracellular Flow Cytometry analysis of C6 (Rat glial tumor glial cell) cells labeling SOX10 with purified ab227680 at 1/20 dilution (3.75µg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue. 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse breast tissue sections labeling SOX10 with ab227680 at 1/100 dilution (0.25 μg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10 mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on the mouse breast, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab227680 for 10 mins at room temperature.

Immunocytochemistry/ Immunofluorescence analysis of B16-F0 (mouse melanoma epithelial cell-like) cells labeling SOX10 with purified ab227680 at 1:25 (3 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Intracellular Flow Cytometry analysis of B16-F0 (Mouse melanoma epithelial cell-like) cells labeling SOX10 with purified ab227680 at 1/200 dilution (0.375µg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue. 

Intracellular Flow Cytometry analysis of A-375 (Human malignant melanoma epithelial cell) cells labeling SOX10 with purified ab227680 at 1/200 dilution (0.375µg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue. 

Immunocytochemistry/ Immunofluorescence analysis of A-375 (human malignant melanoma epithelial cell) cells labeling SOX10 with purified ab227680 at 1:25 (3 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Formalin-fixed, paraffin-embedded human melanoma tissue stained for SOX10 using ab227680 at 1/100 dilution in immunohistochemical analysis.