Anti-SOX9 抗体 [EPR14335-78] (ab185966)

Western blot: Anti-Sox9 antibody [EPR14335-78] (ab185966) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab185966 was shown to bind specifically to Sox9. A band was observed at 50-70 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in Sox9 CRISPR-Cas9 edited cell line. The band observed in the CRISPR-Cas9 edited lysate lane below 50-70 kDa is likely to represent a truncated form of Sox9. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Sox9 CRISPR-Cas9 edited HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Immunofluorescence staining of SOX9 using ab185966 in primary hippocampal mouse neurons/glia, (obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP), DIV14. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% TritonX-100 (in PBS) for 5 mins 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 ab185966 at 1 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green), ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple), all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected, most GFAP positive cells are also SOX9 positive, while NeuN positive cells are SOX9 negative. SOX9 positive cells, which are not GFAP positive (e.g. asterisk) are likely neural stem cells/ oligodendrocyte precursor cells present in the culture.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

ab185966 staining SOX9 in developing eye of mouse tissue section by Immunohistochemistry (PFA perfusion fixed frozen sections).

Tissue samples were fixed by perfusion with formaldehyde, cut into 20 micron slices, blocked with 2% BSA for 10 minutes at 21°C and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/1000 in PBS) at 21°C for 4 hours. A biotin-conjugated goat anti-rabbit polyclonal (1/300) was used as the secondary antibody.

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Blocking and diluting buffer: 5% NFDM/TBST

SOX9 can be ubiquitinated or SUMOylated to higher molecular weight (PMID: 24155239, PMID: 16307912, PMID: 16554309, PMID: 32070068). Meanwhile, it has a truncated version as mini-Sox9 (PMID: 21297661,PMID: 27429045).

HeLa expresses very low level of SOX9 (PMID: 18296708, PMID: 18677406).

ab185966 staining Sox9 in F9 (Mouse embryonic testicular cancer cell line) cells.

The cells were fixed with 4% formaldehyde (10 min), permeabilized 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 ab185966 at a 5 μg/ml concentration and ab7291, mouse monoclonal [DM1A] to alpha Tubulin, at  1μg/ml concentration, followed by a further incubation at room temperature for 1 h with an anti-rabbit AlexaFluor^® 488 (ab150081) at 2 μg/ml (shown in green) and an anti-mouse AlexaFluor^® 594 (ab150120) at 2 μg/ml (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Intracellular Flow Cytometry analysis of PC-3 (human prostate adenocarcinoma) cells labeling SOX9 with purified ab185966 at 1/120 (red). 

Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

Immunohistochemistry analysis of paraffin-embedded rat colon tissue labeling SOX9 with ab185966 at 1/1000 dilution. Counterstained with hematoxylin.

Immunofluorescence analysis of 4% paraformaldehyde fixed PC-3 (Human prostate adenocarcinoma cell line) cells labeling SOX9 with ab185966 at 1/250 dilution (Left panel, red).

Goat anti Rabbit IgG (Alexa Fluor^®555) used as secondary antibody at 1/200 dilution. DAPI staining (Right panel, blue).

Immunohistochemistry analysis of paraffin-embedded mouse colon tissue labeling SOX9 with ab185966 at 1/1000 dilution. Counterstained with hematoxylin.

Immunohistochemistry analysis of paraffin-embedded human colon tissue labeling SOX9 with ab185966 at 1/1000 dilution. Counterstained with hematoxylin.

Immunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue labeling SOX9 with ab185966 at 1/1000 dilution. Counterstained with hematoxylin.

Immunohistochemical analysis of Formalin/PFA-fixed paraffin-embedded pig small intestine sections labeling SOX9 with ab185966 at dilution of 1/2000.

The secondary antibody used was a polyclonal goat anti-rabbit biotin conjugated antibody at a dilution of 1/300. The sample was counterstained with hematoxylin. Antigen retrieval was heat mediated using citric acid.

The image shows intense enterocyte/goblet cell nuclear positivity, confined to the crypts of Lieberkühn.

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Immunofluorescence analysis of 4% paraformaldehyde fixed SW480 (Human colorectal adenocarcinoma cell line) cells labeling SOX9 with ab185966 at 1/250 dilution. Goat anti Rabbit IgG (Alexa Fluor^®555) used as secondary antibody at 1/200 dilution. DAPI staining shown in blue.