Mice were anesthetized using pentobarbital sodium (50 mg/kg), perfused with saline, followed by 4% methanol in 0.1 M phosphate buffer (PBS). After which brain tissue was removed, postfixed into 4% methanol for 6 h, and then immersed in 30% sucrose. After 3 days, a freezing microtome (Leica) was used to obtain 40-μm-thick coronal brain sections. Slices were then washed with PBS and treated with 1% Triton-100, followed by goat serum and incubation with c-fos primary antibody (Santa Cruz Biotechnology, -sc-52, 1:3,000) at 4°C overnight. Slices were then incubated with the corresponding fluorescence-conjugated secondary antibody [Alexa Fluor 488 (1:500, A11034, Invitrogen)] at room temperature for 2 h. Images with fluorescence were captured using a Nikon A1 confocal microscope with the resolution of 1,024 × 1,024 pixels and were then processed using National Institutes of Health (NIH) ImageJ software.
C fos primary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
The C-fos primary antibody is a tool used in scientific research to detect the presence and levels of the c-Fos protein, which is an important transcription factor involved in cellular processes. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of c-Fos in different biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Freezing microtome, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Fp1020, by PerkinElmer (1 mentions)
Vectastain abc, by Vector Laboratories (1 mentions)
Protein a g plus agarose beads, by Thermo Fisher Scientific (1 mentions)
Cellytic nuclear extraction kit, by Merck Group (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions)
Donkey anti rabbit biotin, by Jackson ImmunoResearch (1 mentions)
6 protocols using c fos primary antibody
1
Immunostaining of c-Fos in Mouse Brain
2
Measuring Fos-like Immunoreactivity in Mouse CeA
3
c-Fos Immunohistochemistry in Stxbp1 Mice
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Stxbp1cre/+, congenic BL6 Stxbp1+/− mice and controls (n = 4, 5 and 5, respectively) were sacrificed by cervical dislocation. Brains were fixed in 4% PFA (paraformaldehyde) and cryoprotected in a 30% sucrose solution overnight. Brains were blocked in the coronal plane, frozen on dry ice, and sectioned at 50 µm on a cryostat. Coronal brain sections were stained with c-Fos primary antibody (Santa Cruz, sc-52; 1:800/1:500). After overnight incubation, sections were incubated with biotinylated goat anti-rabbit secondary antibody (#65-6140, Invitrogen; 1:400). The sections were then incubated in avidin-biotin peroxidase complex (Vectastain ABC, Vector Laboratories; 1:800) and peroxidase labelling was visualized by DAB/nickel substrate working solution (DAB Peroxidase Substrate, SK-4100; Vector Laboratories). Images were obtained using a Leica light microscope (×10 magnification) and number of c-Fos positive cells was counted using ImageJ software. A detailed description of the protocol and analysis can be found in theSupplementary material .
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Stxbp1cre/+, congenic BL6 Stxbp1+/− mice and controls (n = 4, 5 and 5, respectively) were sacrificed by cervical dislocation. Brains were fixed in 4% PFA (paraformaldehyde) and cryoprotected in a 30% sucrose solution overnight. Brains were blocked in the coronal plane, frozen on dry ice, and sectioned at 50 µm on a cryostat. Coronal brain sections were stained with c-Fos primary antibody (Santa Cruz, sc-52; 1:800/1:500). After overnight incubation, sections were incubated with biotinylated goat anti-rabbit secondary antibody (#65-6140, Invitrogen; 1:400). The sections were then incubated in avidin-biotin peroxidase complex (Vectastain ABC, Vector Laboratories; 1:800) and peroxidase labelling was visualized by DAB/nickel substrate working solution (DAB Peroxidase Substrate, SK-4100; Vector Laboratories). Images were obtained using a Leica light microscope (×10 magnification) and number of c-Fos positive cells was counted using ImageJ software. A detailed description of the protocol and analysis can be found in the
4
Quantifying NLRP3 Gene Expression in HMC3 Cells
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Nucleoprotein in HMC3 cells was extracted with a CelLytic™ NuCLEAR™ Extraction Kit (Sigma-Aldrich; Merck KGaA, Germany) and incubated c-Fos primary antibody (1:500 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C for 60 min with gentle mixing. Subsequently, 20 µl Protein A/G Plus-Agarose beads (Thermo Fisher Scientific, Inc.) were added, followed by incubation at 4 °C overnight. The mixture was centrifuged at 500 x g for 5 min at 4 °C. The supernatant was discarded and the Co-IP products were washed three times with PBS. After the final wash, the precipitates were re-suspended in 40 μl sample buffer and detected by PCR to quantify the NLRP3 gene expression.
PCR was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) with SYBR Premix Ex Taq II (Takara). The PCR primer sequences for NLRP3 and GAPDH were as follows: NLRP3, (F)5'-GCTGGTCTTGAATTCCTCA-3' and (R) 5'-GGCACACGGATGAGTCTTT-3'; GAPDH, (F) 5'-TACTAGCGGTTTTACGGGCG-3' and (R) 5'- TCGAACAGGAGGAGCAGAGAGCGA-3'. A melting curve analysis of the amplified products was performed at the end of each PCR cycle. The comparative C(T) method was used to quantify the expression of NLRP3 using GAPDH as the normalization control.
PCR was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) with SYBR Premix Ex Taq II (Takara). The PCR primer sequences for NLRP3 and GAPDH were as follows: NLRP3, (F)5'-GCTGGTCTTGAATTCCTCA-3' and (R) 5'-GGCACACGGATGAGTCTTT-3'; GAPDH, (F) 5'-TACTAGCGGTTTTACGGGCG-3' and (R) 5'- TCGAACAGGAGGAGCAGAGAGCGA-3'. A melting curve analysis of the amplified products was performed at the end of each PCR cycle. The comparative C(T) method was used to quantify the expression of NLRP3 using GAPDH as the normalization control.
5
Immunofluorescent Staining of c-Fos
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After treatment, cells were fixed in 4% paraformaldehyde at room temperature and permeabilized with PBS/Triton 0.5%. Cells were stained overnight at 4°C with c-fos primary antibody (Santa Cruz Biotechnology), as described in Supplemental Experimental Procedures . Nuclei were counterstained with DAPI.
6
Immunohistochemical Staining of c-Fos
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