Name: the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo
Uploaded: 2016-04-25T14:00:00
Description: Watch this Scientific Journal Video about The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The University Paris 13 supported this work. ASPT was supported by a University Paris 13 fellowship and the "Association Fran&#231;aise pour le Syndrome d'Ondine". FJ was supported by a Laboratory of Excellence GR-Ex fellowship. The GR-Ex (ref ANR-11-LABX-0051) is funded by the program "Investissement d'avenir" of the French National Research agency (ref ANR-11-IDEX-0005-02).

Note: c-FOS detection is a standardized procedure involving several steps (Figure 1). All experiments were performed on rats or mice. Experimental protocols were approved by the Ethics Committee in Animal Experiment Charles Darwin (Ce5/2011/05), done in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU) for animal care, and conducted in accordance with French laws for animal care.
1. Preparation of Solutions
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	<li>Prepare 0.2 M ...

<ol>
	<li>Curran, T., Teich, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+39,000-dalton+protein+in+cells+transformed+by+the+FBJ+murine+osteosarcoma+virus.">Identification of a 39,000-dalton protein in cells transformed by the FBJ murine osteosarcoma virus.</a> Virology. 116, 221-235 (1982).</li><li>Curran, T., MacConnell, W. P., van Straaten, F., Verma, I. 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C-fos is an immediate early gene, and the detection of its product, the c-FOS protein, is classically used to identify neuronal populations involved in specific respiratory responses in vivo11,13,25,28 and ex vivo16-18,27,32,33.
Critical Steps Within the Protocol
Be careful during the perfusion step. The 4% PFA solution must be well prepared and the fixation and post-fixation steps m...

The c-fos gene was identified for the first time at the beginning of 19801,2 and its product was characterized in 1984 as a nuclear protein having gene-activator properties3,4. It participates in long-term mechanisms associated with neuron stimulation. Indeed, changes in&#160;neuronal activity lead to second messenger signaling cascades that induce the expression of the immediate early gene c-fos, which induces the production of the transcription factor c-FOS. The latter initiates the expression of late genes and thus participates in&#160;adaptive responses of the nervous system to many different types of stimuli4. Thus, since the end of 19805,6, c-FOS protein detection has been frequently used to study the effects of exogenous factors on gene transcription in general4 and on the activity of the central nervous system (CNS) for mapping out neuronal pathways involved in different physiological conditions.
Basal c-fos expression has been studied in various species including mice, rat, cat, monkey, and human4. Thereby, the kinetics of its expression is relatively well known. The transcription activation is rapid (5 to 20 min)7,8, and the mRNA accumulation reaches a maximum between 30 and 45 min after the onset of stimulation9 and declines with a short half-life of 12 min. The c-FOS protein synthesis follows mRNA accumulation and could be detected by immunohistochemistry at&#160;20 to 90 min&#160;post stimulation6.
Analysis of c-fos expression is classically used in in vivo studies to identify the central respiratory network involved in the ventilatory responses to hypoxia or hypercapnia10-14. More recently, this tool was also used in ex vivo brainstem preparations to explore central respiratory network adaptations to hypoxia or hypercapnia15-18. Indeed, these preparations generate a rhythmic activity classically assimilated to the central respiratory drive19. Thus, this type of preparation has the advantage of being completely deafferented, and therefore, results regarding c-fos expression only reflect the consequences of a central stimulation without any intervention of peripheral structures.
The c-FOS detection could be made by immunohistochemical or immunohistofluorescence approaches. Indirect immunodetection necessitates the use of a primary antibody against c-FOS and a secondary antibody directed against the species in which the primary antibody was produced. For the immunohistochemical method, the secondary antibody is conjugated with an enzyme (peroxidase, for example) that acts on a substrate (H2O2 for the peroxidase). The product of the enzymatic reaction is developed by a chromogen (3.3-diaminobenzidine tetrahydrochloride), which stains it and can be observed under light microscopy. The reaction could be reinforced using nickel ammonium sulphate. These methods allow the detection of actives neurons during different physiological challenges and therefore the identification and/or the mapping of peripheral and central pathways involved in the consecutive physiological responses.

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			<td height="21" style="height:21px;width:216px;">Cell culture plate 12-Well</td>
			<td style="width:71px;">Costa</td>
			<td style="width:119px;">35/3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:107px;width:216px;">15 mm Netwell inserts with mesh polyester membrane</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">3477</td>
			<td style="width:167px;">The 15mm diameter well inserts have 74&#181;m polyester mesh bottoms attached to polystyrene inserts</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">primary antibody (rabbit polyclonal antibody against the c-Fos protein)</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-52</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vectastain Elite ABC KIT&#160;</td>
			<td rowspan="2" style="width:71px;">Vector laboratories</td>
			<td rowspan="2" style="width:119px;">PK-6101</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(Rabbit IgG-secondary antibody)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaH2PO4*2H2O</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">71505</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH 0.1N</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">43617</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyvinyl-Pyrrolidone</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PVP-360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7903</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene-glycol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">33068</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X100</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisma HCl</td>
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</table>

the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS detection is a useful tool that allows identifying groups of activated cells under specific conditions such as hypoxia and hypercapnia in vivo (Figure 2A) or in situations that mimic&#160;these conditions ex vivo (Figure 2B). In vivo, newborn, young, or adult rodents were placed in an airtight box in which the gaseous environment is continually renewed by a gas mixture with a composition precisely defined for 30 to 180...

Watch this Scientific Journal Video about The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

The overall goal of this immunohistochemical detection against c-FOS protein is to assess the neuronal activity in brain tissues. This method can help answer our key questions in the neurophysological field such as brain mapping and identification of brain regions involved in specific physiological regulations. The main advantage of this technique is that c-FOS gene has a low expression in the absence of stimulation, which allows easier quantification of neuronal activity in our test simulation.To begin this procedure, wash the coronal brain stem sections for 10 minutes with 0.1 molar PBS at room temperature. Repeat the wash three times. Next, suppress the endogenous peroxidase activity by incubating the sections with 3%hydrogen peroxide in 0.1 molar PBS for 30 minutes at room temperature.Then, wash the sections for 10 minutes with 0.1 molar PBS at room temperature and repeat three times. Block nonspecific binding sites by incubating the coronal sections with 2%goat serum in PBST for one hour at room temperature. Subsequently, incubate the sections with a rapid polyclonal antibody against the c-FOS protein in PBST with 0.25%bovine serum albumin for 48 hours at four degrees Celsius.In this step, wash the sections with 0.1 molar PBS for 10 minutes at room temperature and repeat the procedure three times. Next, incubate the coronal sections for one hour at room temperature with a biotinylated goat anti-rabbit antibody in PBST with 0.25%bovine serum albumin. After one hour, wash the sections for 10 minutes with PBST at room temperature and repeat three times.Then, incubate the coronal sections for one hour with an avidin biotin peroxidase complex in PBST. Subsequently, wash the sections with PBST at room temperature for 10 minutes and repeat two more times. Then, wash the sections for 10 minutes with 05 molar Tris buffer at room temperature and repeat the wash two more times.Next, incubate the coronal sections with DAB, nickel ammonium sulfate, and hydrogen peroxide in 05 molar Tris buffer at room temperature. Add 17 microliters of hydrogen peroxide to the remaining Tris buffer. When the staining intensity is optimal, stop the reaction by washing the sections with 0.1 molar PBS for 10 minutes at room temperature, and repeat the wash four times.Then, wash the sections with distilled water. Now, mount the sections serially on the slides by carefully spreading them in rostrocaudal order on the slides with a brush. After that, let the sections air dry and clearly label the slides according to the samples.Then, dehydrate the sections by immersing the slides in an absolute alcohol bath for 30 seconds at room temperature, and repeat the procedure twice. Next, immerse the slides in a xylene bath for three minutes at room temperature and repeat twice. Subsequently, apply five drops of mounting medium on each slide.Place cover glass on them and avoid forming air bubbles. Let the slides air dry for 48 hours. Shown here is a drawing of a section of the medula oblangata.The dotted area shows the commissural and median parts of the nucleus tractus solitarius. The rectangle indicates the region that photomicrographs were taken. These are the photomicrographs of c/mNTS in the condition of normoxia or under hypoxia.The black arrows show c-FOS-positive cells. Here is a histogram showing the mean number of c-FOS-positive cells per section in c/mNTS in the condition of normoxia or under hypoxia. The number of c-FOS-positive cells was significantly higher in the condition of hypoxia than that of normoxia.After watching this video, you should have a good understanding of how to use the c-FOS immunohistochemical detection as a marker of central pathway involved in specific physiological stimulation.

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