For quantification of c-fos immunoreactivity after social contacts, mice were individually housed on the day before testing day. On the testing day, a stranger male juvenile mouse (social stimulus) was introduced in the cage and allowed to interact with the experimental mouse for 10 min. The experimental mice were transcardially perfused with 4% PFA 30 min after introducing the social stimulus. Brains were extracted and post-fixed overnight in 4% PFA. Coronal sections (50 μm) were immunostained using a rabbit anti-c-fos antibody (1:5000; Cell Signaling Technology) applied overnight in a PBS solution containing 0.3% Triton X-100 (PBS-T), at room temperature. Sections were then washed in PBS-T and incubated in a 1:500 dilution of goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibodies, Alexa Fluor Plus 488 or 555 (Thermo Fisher Scientific) in PBS-T for 1 h. The sections were rinsed with PBS-T and mounted using mounting medium. Images were acquired using a confocal microscope (Olympus FluoView FV1200) and quantitatively analyzed with ImageJ.
Rabbit anti c fos antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit anti-c-Fos antibody is a laboratory tool used for the detection and analysis of the c-Fos protein in biological samples. c-Fos is a transcription factor that plays a role in cellular signal transduction pathways. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to identify and quantify the presence of c-Fos in various cell and tissue types.
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Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluoview fv1200, by Olympus (1 mentions)
Diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Vibratome, by Leica (1 mentions)
Biotinylated goat anti rabbit, by Vector Laboratories (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Donkey anti rabbit cy3 antibody, by Jackson ImmunoResearch (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
0.01 m pbs, by Merck Group (1 mentions)
Muscimol, by Merck Group (1 mentions)
Goat serum, by Jackson ImmunoResearch (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Naloxone, by Merck Group (1 mentions)
Rabbit anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Fluoroshield medium with dapi, by Abcam (1 mentions)
Cm1850 cryostat, by Leica (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
9 protocols using rabbit anti c fos antibody
1
Quantifying c-fos Immunoreactivity After Social Interaction
2
c-Fos Immunohistochemistry in Mouse Brain
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Vehicle or CNO-treated CamKIIα-tTA:TetO-hM3Dq adult male mice, naive for behavioural testing, were sacrificed by transcardial perfusion with 4% paraformaldehyde 2 h post-drug treatments, for c-Fos immunohistochemistry (IHC). Free-floating (40 μM) coronal sections cut on the vibratome (Leica, Germany) were incubated with the blocking solution (10% horse serum, 0.3% TritonX-100 in 0.1M Phosphate buffer) at room temperature for 2 h. Sections were then incubated with rabbit anti-c-Fos antibody (1:1000, Cat no. 2250, Cell Signalling Technology, United States) for 2 days at 4°C, followed by sequential washes, and incubation with the secondary antibody (biotinylated goat anti-rabbit, 1:500, Cat no. BA9400, Vector Labs, United States) for 2 h at room temperature. Signal amplification was performed using an Avidin-biotin complex based system (Vector lab, Vectastain ABC kit Elite PK1600, United States) and visualised using the substrate, Diaminobenzidine tetrahydrochloride (Cat no. D5905, Sigma-Aldrich, United States).
3
Immunofluorescent Labeling of Histamine and c-Fos in Mouse Brain
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Mice were anaesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (25 mg/kg, i.p.) and perfused with 60 ml saline, followed by 70 ml 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were removed and immersed in the same fixative for 24 hours at 4 °C, and the solution was replaced with PB containing 30% sucrose and 0.1% sodium azide for 48 hours. Then, the brains were frozen in O.C.T. compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan). The brains were coronally sectioned (30 μm thickness) on a freezing cryostat. For HDC and c-fos double-staining, sections were incubated with a goat anti-HDC antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-c-fos antibody (Cell Signaling Technology, Beverly, MA) diluted 1:500 in PB saline containing 0.3% Triton X-100 (PBS-T) and 0.1% sodium azide for 72 hours at 4 °C. These sections were incubated with the Alexa 594-labelled donkey anti-goat IgG antibody or Alexa 488-labelled donkey anti-rabbit IgG (1:500, Life Technologies, Gaithersburg, MD) for 2 hours at room temperature. The sections were mounted on glass slides and examined with a fluorescence microscope (DM4000, DFC550, Leica Japan, Tokyo, Japan).
4
Immunohistochemical Analysis of c-Fos Expression in the Arcuate Nucleus
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Coronal sections throughout the rostrocaudal axis of the ArcN were collected on a cryostat at a thickness of 40 μm, washed in PBS 3 × 10 min, and placed into a blocking solution containing PBST (PBS +0.1% Triton X‐100) and 5% v/v normal donkey serum for 2 h at room temperature. Next, a rabbit anti‐c‐fos antibody (1:500; Cell Signaling Technology) was added to the blocking solution, and sections were then incubated overnight at 4°C under gentle agitation. On the next day, sections were washed 3 × 10 min in PBST, and an AlexaFluor568 conjugated donkey anti‐rabbit IgG secondary antisera (1:500; Abcam) was added for incubation for 2 h at room temperature. Finally, sections were washed 3 × 10 min in PBST followed by a 1 × 10 min wash in PBS, mounted onto microscope slides, coverslipped using Vectashield antifade mounting medium (Vector Laboratories), and sealed with clear nail polish.
5
Amylin-Induced c-Fos Expression in Rat Brain
6
Opioid Receptor Signaling Pathway Protocol
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Morphine was purchased from Shenyang No.1 Pharmaceutical Factory, China. 0.01 M PBS, Triton X-100, naloxone and muscimol were purchased from Sigma, United States. Rabbit anti-c-Fos antibody (Cat#5348) was purchased from Cell Signaling Technology, United States. Goat serum and Alexa Fluor 594-conjugated goat anti-rabbit antibody were purchased from Jackson Immuno Research Laboratory, United States. Biotinylated anti-rabbit secondary antibody was purchased from Vector Laboratories, United States. Other reagents in artificial cerebrospinal fluid (ACSF) were the products of Shanghai Chemical Plant, China.
7
Immunohistochemical Analysis of Brain Tissue
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A different group of mice was used for immunohistochemistry studies. After 9 h of fasting or ad libitum, the mice were anesthetized with 1.4% isoflurane for 3 min and perfused transcardially with PBS followed by 4% cold, buffered paraformaldehyde. The brain tissues were removed and post-fixed at 4°C overnight, and then cryoprotected in 15% sucrose followed by 30% sucrose in PBS for 48 h. 20-μm coronal sections were obtained with a Leica CM1850 cryostat. Non-specific antibody binding was inhibited by incubating the slices in 0.1 M PBS containing 10% goat serum and 0.3% Triton X-100. Slices were then incubated for 24 h at 4°C with one of the following primary antibodies: rabbit anti-c-Fos antibody (#2250, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-caspase-3 antibody (#9662, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States), and rabbit anti-cleaved caspase-3 antibody (#9661, 1:200 dilution, Cell Signaling Technology, Danvers, MA, United States). After the incubation, tissue sections were washed and incubated for 1 h at room temperature with Alexa Fluor 488 or Alexa Fluor 594 (1:500 dilution, Thermo Fisher Scientific, Rockford, IL, United States) as the secondary antibodies. Fluoroshield medium with DAPI (ab104139, Abcam, Cambridge, MA, United States) was used for nuclear counterstain.
8
Immunofluorescent Analysis of c-Fos in STN
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After deeply anesthetized with 1% pentobarbital sodium (50 mg/kg), the mice were perfused transcardially with PBS followed by 4% paraformaldehyde. The right STN tissues were extracted and further fixed using 4% paraformaldehyde at 4°C for 12 h. Then the tissues were cryoprotected with 30% sucrose and embedded in OCT compound. The STN slices (30 μm) were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) and blocked with 5% bovine serum albumin (BSA, BioFroxx, Germany) for 1 h at room temperature. The sections were incubated for 24 h at 4°C with rabbit anti-c-Fos antibody (1:500, No. 2250, Cell Signaling Technology, Danvers, MA, United States). After washing with PBS three times for 15 min, the slices were incubated with secondary DyLight® 594 donkey anti-rabbit IgG (1:400, EarthOx, Burlingame, CA, United States) for 1 h at room temperature. Images were captured using LSM780 laser confocal scanning microscope (Zeiss, Oberkochen, Germany). The mean fluorescence intensity was calculated using ImageJ software for quantitative analysis of the expression level of c-Fos in STN neurons.
9
Whole-Brain Imaging of c-fos Expression
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Male C57BL/6JRj mice (8 weeks; JanVier, Le Genest-Saint-Isle, France) received a single administration of ulotaront (3 mg/kg, ip), RO5263397 (0.3 mg/kg, ip) or the respective vehicle 105 min prior to the transcranial perfusion (heparinized PBS and 10% neutral buffered formalin (NBF)). Subsequently, the brains were dissected and postfixed overnight in 10% NBF at room temperature. The iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) [31 (link),32 (link)] protocol was used for whole brain immunolabelling as previously described [33 (link),34 (link)]. For visualization of c-fos expression, rabbit anti-c-fos antibody (1:5000, Cell Signaling Technology) was used followed by incubation with the secondary donkey anti rabbit Cy-5 antibody (1:1000, Jackson ImmunoResearch). The optically transparent brain samples were imaged using LaVision ultramicroscope II (Miltenyi Biotec). Image processing, registration and cell detection was performed according to the method of Perens and colleagues [33 (link)]. In addition to voxel-level analysis, a total of 839 brain regions of interest (ROIs) were analyzed.
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