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Anti c fos antibody

Manufactured by Abcam
Sourced in United States

Anti-c-fos antibody is a laboratory tool used to detect the presence and quantify the levels of the c-Fos protein in biological samples. c-Fos is an immediate early gene that is rapidly induced in response to various cellular stimuli, making it a widely used marker for neuronal activation.

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7 protocols using anti c fos antibody

1

Osteoclastogenesis Induction Protocol

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Recombinant mouse soluble RANKL (Gene ID: 21943) was obtained from R&D Systems (Catalog number: 462-TEC-010, Oakville, ON, Canada). Dulbecco modified Eagles medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/mL), and TRIzol Reagent were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). Ovotransferrin (conalbumin from chicken egg white) with purity ≥98% was purchased from Sigma-Aldrich (Catalog number: C0755, St. Louis, MO, USA). The tartrate-resistant acid phosphatase (TRAP)-staining kit was obtained from Sigma-Aldrich (Catalog number: 387A-1KT, St. Louis, MO, USA). The annexin V-FITC Apoptosis Staining/Detection Kit was purchased from Abcam (Catalog number: ab14085, Toronto, ON, Canada). The NF-κB pathway sampler kit (Catalog number: 9936T), MAPK family antibody sampler kit (Catalog number: 9926T), and phosphor-MAPK family antibody sampler kit (Catalog number: 9910T) were purchased from Cell Signaling Technology (Whitby, ON, Canada). Recombinant anti-TRAF6 antibody (Catalog number: ab33915), anti-c-Fos antibody (Catalog number: ab190289), and anti-α tubulin antibody (Catalog number: ab7291) were purchased from Abcam (Cambridge, MA, USA). NFATc1 antibody (Catalog number: 7A6) and cathepsin K antibody (Catalog number: E-7) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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2

Immunohistochemical Analysis of Spinal Cord Pain Signals

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Mice were anesthetized using sodium pentobarbital (60mg/kg, i.p. injection), and perfused intracardially with 0.9% saline followed by 4% formaldehyde. The spinal cord of L4–5 was removed and post-fixed in 4% paraformaldehyde for 3 h at room temperature. It was the equilibrated in 30% sucrose in phosphate buffer overnight at 4 °C. Thirty micrometer transverse series sections were cut on a cryostat and stored in phosphate buffer. For immunofluorescence staining, free-floating sections were blocked in TBS containing 5% donkey serum at room temperature for 2 h, and then incubated in primary antibody at 4 °C overnight. Sections were then washed in PBS (3 times for 5 min each), followed by incubation in secondary antibody at room temperature for 2 h and additional washing. Sections were mounted on slides and covered with 90% glycerin, and then observed under a confocal microscope (FluoView FV1000; Olympus). The anti-c-fos antibody (Abcam) was diluted 1:200. Quantification of immunohistochemical results was done for three sections from spinal cord dorsal horn layer I, II, V, each of which are associated with pain (n = 8 mice per group). Sections were taken from the region within lumber segments L3–L5.
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3

Immunostaining of c-Fos in Brain Tissue

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Brains were cut on a Leica CM3050S cryostat at 30 uM and stored in 1:1 TBS/glycerol solution at −20°C until ready to stain. To stain, slices were first washed in 0.2% Triton-X in Tris-buffered saline (TBS) solution 3 times × 10 minutes. Slices were then blocked for 1 hour in solution of 0.3% Triton-X and 5% normal goat serum (NGS) in PBS. Slices were stained overnight in 0.3% Triton-X, 5% NGS, and 1:20,000 anti-c-fos antibody (Abcam - ab190289) with gentle shaking at 4°C. Slices were then washed 3 times in solution of 0.3% Triton-X and 5% NGS in PBS (10 minutes first wash, 30 minutes second wash, 40 minutes third wash). Slices were then stained with secondary antibody (AlexaFluor 488, Invitrogen – A-11034) in 0.3% Triton-X, 5% NGS, and 1:200 secondary antibody for 2 hours. Slices were then washed in 1X PBS (10 minutes first wash, 30 minutes second wash, and 40 minutes third wash), with DAPI (R&D Systems 5748) added at a dilution of 1:10,000 for last 10 minutes of second wash. Slices were mounted on VWR Superfrost® Plus microscope slides with a drop of Vecta-Shield anti-fade medium (Vector Laboratories 101098-042).
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4

Immunohistochemical Analysis of c-Fos and Parvalbumin in Mouse Brain Tissue

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As previously described (Han et al., 2020 (link)), mice were anesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS), and tissues were fixed in 4% PFA at 4°C for 12 h. After dehydration by 30% sucrose, brain tissue was cut into 30-μm-thick sections using a freezing microtome (Leica CM30505, Germany). The posterior portion of the BLA-containing sections was permeabilized with 0.3% Triton X-100 and 5% donkey serum in PBS for 2 h and then incubated with rabbit polyclonal anti-c-Fos antibody (1:1,000, Abcam, Cambridge, United Kingdom, ab190289) or rabbit polyclonal anti-PV antibody (1:1,000, Abcam, ab11427) at 4°C overnight. After washing three times with PBS, sections were incubated with Cy3-labeled anti-rabbit secondary antibody (1:1,000, Absin, Shanghai, China, abs20024) for 1.5 h at room temperature and then given three 10-min washes in PBS. Afterward, sections were incubated with DAPI solution (1:5 for 15 min, Beyotime, Shanghai, China, C1005) for nuclear labeling and coverslipped with anti-fade mounting medium. Images were captured using an EVOS fluorescence microscope (Invitrogen, United States). For c-Fos immunostaining, mice subjected to several behavioral tests were perfused 1.5 h after the tests.
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5

Antibody and ELISA Reagent Procurement

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Antibodies against GFAP and amyloid beta (Aβ) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-c-Fos antibody was purchased from Abcam (Cambridge, MA, USA). Anti-Iba-1 antibody was purchased from Wako Chemicals USA, Inc. (Richmond, VA, USA). Elite Vectastain ABC reagents, Vector VIP, biotinylated anti-rabbit, and anti-mouse antibodies were purchased from Vector Laboratories Inc (Burlingame, CA, USA). The Aβ 1–40 and Aβ 1–42 enzyme-linked immunosorbent assay (ELISA) kits were obtained from EMD Millipore (Burlington, MA). Standards used in mass spectroscopy analysis were purchased from Millipore-Sigma Burlington, MA, USA. Autosampler vials were obtained from ThermoFisher Scientific, (Waltham, MA, USA), Silanized micro-vial inserts from Agilent (Santa Clara CA; part #5181–8872) and inserts were from VWR (Radnor, PA, USA).
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6

Immunohistochemical Evaluation of Transcription Factors in Lung and Colon

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IHC assay was carried out to assess activating transcription factor 2 (ATF2), c-jun, and c-fos expressions in lung and colon tissues. Briefly, 6 µm lung or colon sections were de-paraffinized in xylene solution and hydrated in a decreasing gradient ethanol solution. Sections were then treated with citrate antigen retrieval solution (Beyotime Biotechnology, 20 min, 95 ℃), permeabilized using 0.5% Triton X-100 solution (20 min), incubated with 3% H2O2 solution, and coated with 3% bovine saline albumin (BSA, Beyotime Biotechnology). Sections were then treated with anti-ATF2 antibody (1:250), anti-c-jun antibody (1:250), and anti-c-fos antibody (1:250, Abcam Biotechnology, MA, USA) overnight at 4 ℃ and HRP-conjugated secondary antibody for 60 min. Relative expressions were visualized using a diaminobenzidine (DAB) solution (Beyotime Biotechnology) and the cell nucleus was stained using hematoxylin solution. Results were observed under a microscope (Nikon, Japan).
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7

Western Blot Analysis of P-gp, c-fos, and Actin

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The cells were harvested and homogenized in RIPA lysis buffer (Sigma-Aldrich). The concentrations of the protein samples were determined by the Bradford method. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10% gel) and transferred to membranes. After blocking with 5% skim milk for 1 h, the membrane was incubated with anti-P-gp antibody (Abcam), anti-c-fos antibody (Abcam) and anti-actin antibody (Sigma-Aldrich) at 4 °C overnight. The next day, the membranes were washed three times with Tris-buffered Saline-Tween 20 (TBST), and incubated with the secondary antibodies (Proteintech Group) for 1 h. After washing with TBST, the target proteins were visualized and subjected to image analysis.
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