Anti-c-Fos antibody [EPR21930-238]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Immunohistochemical analysis of formalin fixed paraffin embedded human bladder cancer labelling c-Fos with ab222699 at a concentration of 0.5 µg/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, pH 8.5 for 32mins.

ab222699 anti-c-Fos [EPR21930-238] 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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permebalized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line grown in serum free medium for 36 hours, followed by addition of 20% FBS for 2 hours (Red) / Untreated control (Green) labeling c-Fos with ab22699 at 1/500 dilution compared with a Rabbit monoclonal IgG (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution.

Serum starvation followed by FBS treatment induces the expression of c-Fos (PMID : 14981092).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Immunohistochemical analysis of paraffin-embedded human bladder carcinoma tissue labeling c-Fos with ab222699 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in tumor cells of human bladder carcinoma (PMID : 28358415). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Immunofluorescnt analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeablised HeLa (human cervix adenocarcinoma epithelial cell) cells labeling c-Fos with ab222699 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cells in serum free medium for 36 hours, followed by addition of 20% fetal bovine serum for 2 hours (PMID : 14981092).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-c-Fos antibody [EPR21930-238] (AB222699)

c-Fos was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) grown in serum free medium for 36 hours, followed by addition of 20% FBS for 2 hours whole cell lysate with ab222699 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab222699 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : HeLa grown in serum free medium for 36 hours, followed by addition of 20% FBS for 2 hours whole cell lysate 10 μg (Input).

Lane 2 : ab222699 IP in HeLa grown in serum free medium for 36 hours, followed by addition of 20% FBS for 2 hours whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab222699 in HeLa grown in serum free medium for 36 hours, followed by addition of 20% FBS for 2 hours whole cell lysate.

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

Exposure time : 30 seconds.

Serum starvation followed by FBS treatment induces the expression of c-Fos (PMID : 14981092).

All lanes:

Immunoprecipitation - Anti-c-Fos antibody [EPR21930-238] (ab222699)

Predicted band size: 41 kDa

Observed band size: 55-60 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Immunohistochemical analysis of paraffin-embedded mouse dentate gyrus tissue labeling c-Fos with ab222699 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic nuclear staining in mouse dentate gyrus (PMID : 24604295). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling c-Fos with ab222699 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining in neurons of mouse cerebral cortex (PMID : 24604295). 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 using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-tissue clearing

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IHC-tissue clearing - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Anti-c-FOS antibody ab222699 was used with Tissue clearing kit – CUBIC (ab316246) and 3D Tissue Staining Kit – CUBIC (ab316248) to penetrate, stain and clear a whole mouse brain. White : nuclear staining, Yellow : c-FOS.

Learn more about tissue clearing kits, reagents, and protocols designed to make it easier to stain whole brains and get more data from each valuable tissue sample.

For a whole mouse brain, we recommend starting with 0.6 ug of ab222699 and using a Fab fragment secondary antibody at 0.4 ug to create an antibody complex before 3D staining (see protocol for details). Additive A was used during the staining process.

The sample was imaged using a light-sheet microscope.

• WB

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Western blot - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Blocking/Dilution buffer : 5% NFDM/TBST.

PMA treatment induces expression of c-Fos, as documented in the literature (PMID : 24386331, PMID : 23300800, PMID : 25695333).

All lanes:

Western blot - Anti-c-Fos antibody [EPR21930-238] (ab222699) at 1/1000 dilution

Lane 1:

Untreated Jurkat (human T cell leukemia cell line from peripheral blood) grown in serum free medium overnight, whole cell lysate at 10 µg

Lane 2:

Jurkat grown in serum free medium overnight, followed by treatment with 200 nM PMA for 4 hours, whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 41 kDa

Observed band size: 55-60 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-c-Fos antibody [EPR21930-238] (AB222699)

Western blot : Anti-c-Fos antibody [EPR21930-238] ab222699 staining at 1/1000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 35-50 kDa in Wild-type A375 cell lysates with no signal observed at this size in FOS knockout A375 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-c-Fos antibody [EPR21930-238] (ab222699) at 1/1000 dilution

Lane 1:

Wild-type A375 cell lysates at 20 µg

Lane 2:

FOS knockout A375 cell lysates at 20 µg

Lane 3:

Wild-type PC-12 ab279978 cell lysates at 20 µg

Lane 4:

FOS knockout PC-12 ab281615 cell lysates at 20 µg

Lane 5:

HeLa cell lysates at 20 µg

Lane 6:

Ramos cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 41 kDa

Observed band size: 35-50 kDa

false