Mice were anesthetized with isofluorane and transcardially perfused with cold 0.1M phosphate buffer, followed by 4% paraformaldehyde (PFA) dissolved in 0.1M PB. Brains were post-fixed for 3 hours in 4% PFA at 4˚C and cryoprotected in 30% sucrose. Frozen brains were sliced into 30 μM sections using a cryostat (CM3050, Leica). Free-floating sections from the whole brain were washed 3x in tris-buffered saline (TBS) and then blocked in 1% bovine serum albumin (BSA) in tritonated TBS (0.25% Triton X-100 in TBS) for 1h. The sections were incubated overnight at room temperature in primary antibody (rabbit anti-cFos antibody (Synaptic Systems, #226003, 1:5000), or rabbit anti-SST antibody (Peninsula Laboratories; #T-4103.0050, 1:1000). Slices were then washed in 1% BSA in tritonated BST, followed by incubation with appropriate secondary antibodies (Alex 568 goat anti-rabbit IgG (H+L) (Life Technologies: #A-11036; 1:500), or Alexa 647 goat anti-rabbit IgG (H+L) (Life Technologies: #A-21245, 1:500). Sections were then mounted with DAPI Fluoromount-G (Southern Biotech).
Rabbit anti c fos antibody
Manufactured by Synaptic Systems
Sourced in Germany
The Rabbit anti-c-Fos antibody is a laboratory reagent used for the detection and analysis of the c-Fos protein, which is a commonly used marker of neuronal activation. This antibody is produced in rabbits and can be used in various immunoassay techniques, such as immunohistochemistry and Western blotting, to identify and quantify the expression of c-Fos in biological samples.
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Lab products found in correlation
Dapi fluoromount g, by Southern Biotech (3 mentions)
Alex 568 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Alexa 647 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Cm3050, by Leica camera (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Zen microscope software, by Zeiss (1 mentions)
Alexa fluor 488 affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Rabbit anti th antibody, by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
Cx41 light microscope, by Olympus (1 mentions)
Image pro premier image analysis software, by Media Cybernetics (1 mentions)
Alexa fluor 647 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Sliding cryostat, by Leica Microsystems (1 mentions)
Fluoromount mounting medium, by Southern Biotech (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Superfrost glass slide, by Thermo Fisher Scientific (1 mentions)
Vs120, by Olympus (1 mentions)
D ap5, by Abcam (1 mentions)
10 protocols using rabbit anti c fos antibody
1
Immunohistochemical Staining of Mouse Brains
2
Circadian Regulation of Hypothalamic Neurons
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POMC-tdTomato mice and NPY-hrGFP mice received injections of either NAMPT (1 mg/kg, i.p.) at 1 p.m. (ZT5) or FK866 (10 mg/kg, i.p.) at 1 a.m. (ZT17). One hour after injection, mice were transcardially perfused with 4% paraformaldehyde (PFA) for 15 min using a peristaltic pump. Whole brains were collected, post-fixed with 4% PFA overnight, and kept in 30% sucrose for 48 h. Brain sections (20 μm) were obtained and incubated overnight at 4 °C with a rabbit anti-c-Fos antibody (1:1000, Synaptic Systems, #226 003). Tissues were rinsed with PBS and then incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, Invitrogen) at room temperature for 1 h. After washing with PBS, the slices were mounted using Vectashield mounting media (Vector Laboratories) and viewed using an LSM 700 confocal microscope (Carl Zeiss). Three matched brain sections, which included the ARC, were analyzed in each animal. The numbers of POMC and NPY/AgRP neurons with nuclear c-Fos immunoreactivity were counted and expressed as a percentage of the total number of POMC or NPY/AgRP neurons. c-Fos positive neurons were counted using ZEN microscope software (version 2.1 blue edition, Carl Zeiss).
3
Immunohistochemical Staining of Mouse Brains
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Mice were anesthetized with isofluorane and transcardially perfused with cold 0.1M phosphate buffer, followed by 4% paraformaldehyde (PFA) dissolved in 0.1M PB. Brains were post-fixed for 3 hours in 4% PFA at 4˚C and cryoprotected in 30% sucrose. Frozen brains were sliced into 30 μM sections using a cryostat (CM3050, Leica). Free-floating sections from the whole brain were washed 3x in tris-buffered saline (TBS) and then blocked in 1% bovine serum albumin (BSA) in tritonated TBS (0.25% Triton X-100 in TBS) for 1h. The sections were incubated overnight at room temperature in primary antibody (rabbit anti-cFos antibody (Synaptic Systems, #226003, 1:5000), or rabbit anti-SST antibody (Peninsula Laboratories; #T-4103.0050, 1:1000). Slices were then washed in 1% BSA in tritonated BST, followed by incubation with appropriate secondary antibodies (Alex 568 goat anti-rabbit IgG (H+L) (Life Technologies: #A-11036; 1:500), or Alexa 647 goat anti-rabbit IgG (H+L) (Life Technologies: #A-21245, 1:500). Sections were then mounted with DAPI Fluoromount-G (Southern Biotech).
4
Immunofluorescence Staining Protocol for Tissue Sections
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For immunofluorescence, frozen sections were washed 3 times for 5 min with 1×PBS before being permeabilized in 0.4% Triton X-100 (Sangon Biotech, A110694) (in PBS) for 15 min. To block the non-specific antibody binding, the sections were incubated in 10% normal goat serum (Absin, abs933) (in PBST) for 1 hour and then incubated with primary antibody (rabbit anti-c-Fos antibody, 1:1000, Synaptic Systems, Cat# 226003; rabbit anti-CGRP antibody, 1:1000, Sigma, Cat# C8198; Alexa-568-conjugated IB4, 1:1000, Thermo Fisher Scientific, Cat# I21412; anti-Tubulin Beta3 antibody, 1:1000, BioLegend, Cat# 657408; rabbit anti-NF200 antibody, 1:1000, Sigma, Cat# N4142; rabbit anti-NeuN antibody, 1:200, Proteintech, Cat# 26975-1-AP; rabbit anti-TH antibody, 1:1000, Millipore, Cat# AB152) overnight at 4 °C. The sections were then washed 3 times for 5 min in PBST and then incubated with the secondary antibody (Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG (H+L), 1:500, Jackson, Cat# 111-545-003; Alexa Fluor 594-AffiniPure Goat Anti-Rabbit IgG (H+L) antibody, 1:500, Jackson, Cat# 111-585-003; Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) preadsorbed antibody, 1:500, Abcam, Cat# ab150083) for 1 hour at room temperature. Next, the sections were washed 3 times for 5 min in PBS and mounted on slides. Images were taken with confocal microscopes (FV3000, Olympus).
5
Quantification of c-Fos and Arc Immunoreactivity
6
cFos Immunohistochemistry in Mouse Brain
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For cFos immunohistochemistry (IHC), mice were deeply anesthetized with pentobarbital (150 mg/kg intraperitoneally, Streuli Pharma, Switzerland) and perfused trans-cardially (4.0% paraformaldehyde, 1X PBS, pH 7.4). Brains were removed, post-fixed (4% PFA overnight), and cryoprotected (30% sucrose, 1X PBS, 4 °C, 48 h). They were then frozen and 40 μm coronal sections were cut with a sliding cryostat (Leica Microsystems, Germany).
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
Subsequently, free floating sections were incubated in blocking solution (1% BSA, 1X PBS, 0.3% TrytonX100) at room temperature for 1 h, followed by incubation with rabbit anti-cFos antibody (1:5000, Synaptic System, Germany, #226 003) in blocking buffer (1% BSA, 1X PBS, 0.1% TrytonX100) overnight at 4 °C under constant shaking. Sections were washed extensively with PBS Tryton 0.1% and then exposed to the secondary antibody (Alexa Fluor 647-conjugated donkey anti-rabbit IgG, Life Technologies, USA) in blocking buffer at room temperature for 2 h. After extensive washing, the sections were incubated with Hoechst (Life Technologies, USA) at 1:1000 in PBS at room temperature for 5 min. Slices were washed extensively with PBS and mounted on superfrost glass slides (ThermoScientific, USA) with Fluoromount mounting medium (SouthernBiotech, USA). Images were acquired on a virtual slide microscope (VS120, Olympus, Japan) with a 10 × objective.
7
Immunostaining of c-Fos in Neurons
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Synaptic Systems provided a rabbit anti-c-Fos antibody (226 003, Synaptic Systems). Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibodies were purchased from Jackson (711-545-152). Jiangsu Heng Rui Pharmaceutical Co. Ltd. Provided dexmedetomidine (Jiangsu, China). Noradrenaline was purchased from Acmec (69815-49-2). TOCRIS provided RS79948 (0987). Tetrodotoxin (TTX) was purchased from Bailingwei J&K (608506). CNQX was purchased from abcam (ab120017). D-AP5 was purchased from abcam (ab144482).
8
Quantifying Neuronal Activity in Looming Response
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To measure neuronal activity of animals exposed to a looming stimulus, we used the immediate early gene, c-Fos, as a marker of neuronal activity. Following the behaviour experiment described above, we transcardially perfused mice with ice-cold 1x phosphate-buffered saline and then with 4% paraformaldehyde. Brains were dissected out, postfixed for 24 hrs at 4°C, cryopreserved in 30% sucrose, and stored at −70°C until subsequent use. To stain for c-Fos protein, we sectioned brains at 40 μm, blocked tissue for 1 hr, and incubated sections for 2 days with rabbit anti-c-Fos antibody (1:4000, Synaptic Systems, 226003). We used donkey anti-rabbit Alexa 647 antibody (1:1000, Invitrogen, A31573) for secondary detection and mounted tissues with DAPI Fluoromount-G (SouthernBiotech, 0100–20). Slides were imaged on an AxioScan.Z1 slide scanner (Zeiss).
9
Glucose-Induced c-Fos Expression in VMH
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Six male Ano4-P2A-Cre/Rosa26-LSL-tdTomato mice (8–12 weeks of age) were used to characterize saline (10 mL/kg, i.p.) or glucose (2 g/kg, i.p.)–induced c-fos in the VMH. Mice were fasted for 2 hours before saline or glucose treatment. Ninety minutes after injection, mice were anesthetized with inhaled isoflurane and quickly perfused with saline followed by 10% formalin. After dehydration with 30% sucrose, the brains were cut into sections of 25 μm. Sections from each mouse were blocked with 3% normal donkey serum for 2 hours and incubated with rabbit anti-c-Fos antibody (1:1,000, 226003, Synaptic Systems) on a shaker at 4°C overnight, followed by the donkey anti-rabbit Alexa Fluor 488 (1:500, A21206, Invitrogen) for 2 hours at room temperature. Slides were cover-slipped and analyzed using a fluorescence microscope. The numbers of c-fos and c-fos/Ano4 double-positive cells in the VMH were counted. Three mice were included in each group for statistical analyses.
10
Immunohistochemical Labeling of c-Fos in Brain
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Brains were permeabilized in PTx.2 / 2.3% (w/v) glycine / 20% DMSO for 48 hours at 37 C and then blocked in PTx.2 / 6% donkey serum / 10% DMSO for 48 hours at 37 C, followed by incubation with the primary antibody: rabbit anti-c-fos antibody (Synaptic Systems, Cat No: 226003; Göttingen, Germany) diluted (1:200) in PBS + 0.2% Tween-20 + 1% (w/v) heparin (PTwH) / 5% DMSO / 3% donkey serum for 120 hours at 37 C. Brains were then washed in PTwH 5X for 1 hour each and incubated with the secondary antibody: Alexa Fluor 647 donkey anti-rabbit antibody (Jackson ImmunoResearch, Cat No: 711–605-152) diluted (1:200) in PTwH / 3% donkey serum for 120 hours at 37 C. After the secondary incubation, brains were again washed in PTwH 5X for 1 hour each.
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