Recombinant Anti-Sp7 / Osterix antibody [EPR21034] (ab209484)

IHC image of SP7/Osterix staining in a section of formalin-fixed paraffin-embedded normal rat E15 embryo performed on a Leica BOND® system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab22552, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of SP7/Osterix staining in a section of formalin-fixed paraffin-embedded normal mouse E15 embryo performed on a Leica BOND® system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab22552, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Anti-Sp7 / Osterix antibody [EPR21034] (ab209484) at 1/1000 dilution + Saos-2 (human osteosarcoma cell line) whole cell lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 45, 47 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Human Sp7 / Osterix has two isoforms (45 and 47 kDa). The 45kDa isoform is predominant (UniProt ID: Q8TDD2). In Saos-2 cells, we observed a faint, extra band at around 37kDa.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse E14.5 developing humerus tissue labeling Sp7 / Osterix with ab209484 at 1/500 dilution, followed by AlexaFluor^® 488 Goat anti-Rabbit (ab150077) at 1/1000 dilution. Positive staining on the mouse embryo E14.5 developing humerus (PMID:27134141) is observed. Counter stained with DAPI.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor^® 488 Goat anti-Rabbit (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat E14.5 rib tissue labeling Sp7 / Osterix with ab209484 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on osteoblasts and chondrocytes of rat E14.5 rib (PMID: 17579353; PMID: 25977369) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse E14.5 rib tissue labeling Sp7 / Osterix with ab209484 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on osteoblasts and chondrocytes of mouse E14.5 rib (PMID: 17579353; PMID: 25977369) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human chondrosarcoma tissue labeling Sp7 / Osterix with ab209484 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on tumor cells of human chondrosarcoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Sp7 / Osterix was immunoprecipitated from 0.35mg of Saos-2 (human osteosarcoma cell line) whole cell lysate with ab209484 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209484 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Saos-2 whole cell lysate 10ug (Input). 

Lane 2: ab209484 IP in Saos-2 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab190908 in Saos-2 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 8 seconds.