Anti-SOX9 antibody [EPR14335]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling SOX9 with ab185230 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-SOX9 antibody [EPR14335] ab185230 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunofluorescent analysis of SW480 cells (4% paraformaldehyd-fixed) labeling SOX9 with ab185230 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor^® 555) secondary at 1/200 dilution and counter-stained with DAPI (blue).

Alexa Fluor^® 488 (ab196450) and Alexa Fluor^® 647 (ab196184) conjugated versions are available for this clone.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SOX9 antibody [EPR14335] (AB185230)

Intracellular flow cytometric analysis of PC3 cells (2% paraformaldehyde-fixed)labeling SOX9 with ab185230 at 1/190 dilution (red) or a rabbit IgG (negative) (green), followed by Goat anti rabbit IgG (FITC) secondary at 1/150 dilution.

Alexa Fluor^®488 (ab196450) and Alexa Fluor^®647 (ab196184) conjugated versions are available for this clone.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-SOX9 antibody [EPR14335] (AB185230)

ab185230 at 1/60 immunoprecipitating SOX9 in SW480 (human colorectal adenocarcinoma) whole cell lysate observed at 56 and 75 KDa (lanes 1 and 2).

The expression pattern observed is consistent with the literature (PMID : 10682837) with an additional off-target band at 46 kD.

Lane 1 (input) : SW480 whole cell lysate 10μg

Lane 2 (+) : SW480 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab185230 in SW480 whole cell lysate

For western blotting, ab185230 (1/1000) was used as the primary antibody and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

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

All lanes:

Immunoprecipitation - Anti-SOX9 antibody [EPR14335] (ab185230)

Predicted band size: 56 kDa

Observed band size: 56 kDa,75 kDa

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Exposure time: 10s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin. Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling SOX9 with ab185230 at 1/2000 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

Heat mediated antigen retrieval was performed with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SOX9 antibody [EPR14335] (AB185230)

Immunofluorescence staining of SOX9 using ab185230 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 ab185230 at 5 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.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• WB

Lab

Western blot - Anti-SOX9 antibody [EPR14335] (AB185230)

Western blot : Anti-Sox9 antibody [EPR14335] (ab185230) 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, ab185230 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.

All lanes:

Western blot - Anti-SOX9 antibody [EPR14335] (ab185230) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Sox9 CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg

Lane 3:

SW480 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 50 kDa,70 kDa

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• WB

Lab

Western blot - Anti-SOX9 antibody [EPR14335] (AB185230)

Blocking/Diluting buffer and concentration : 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).

All lanes:

Western blot - Anti-SOX9 antibody [EPR14335] (ab185230) at 1/1000 dilution

Lane 1:

SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

Mouse colon tissue lysate at 20 µg

Lane 5:

Mouse P0 bone tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 42 kDa,56 kDa,75 kDa

false

Exposure time: 40s

• WB

CiteAb

Western blot - Anti-SOX9 antibody [EPR14335] (AB185230)

SOX9 western blot using anti-SOX9 antibody [EPR14335] ab185230. Publication image and figure legend from Liu, N., Fu, D., et al., 2020, Arthritis Res Ther, PubMed 32398124.

ab185230 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab185230 please see the product overview.

Effects of AA on chondrogenic marker expression of chondrocytes. The cells were treated with or without AA (5 μM) for 3 days. a Representative immunofluorescence images of ACAN (yellow), COL-II (green), and SOX9 (red). Nucleus was stained with DAPI (blue). b The RFU analysis of ACAN, COL-II, and SOX9. Unpaired t test; ns, not significant. c Relative mRNA expression of ACAN, COL2a1, and SOX9. Unpaired t test, *p < 0.05, **p < 0.01. d Representative western blot of ACAN, COL-II, and SOX9. GAPDH was served as a loading control. e Representative Alcian Blue staining of human OA chondrocytes. Each experiment was repeated three times

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