eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence
<ol>
	<li>Prepare solutions including the blocking buffer, antibody buffer, washing buffer 1, and washing buffer 2 (see Table 1).</li>
	<li>Rinse slices once with phosphate buffer (PB) to remove excess optimal cutting temperature (OCT) compound.
	<ol>
		<li>Remove the solution with a plastic pipette without sucking in the brain slices. Add 250 &#181;L of fresh PB with a 1000 &#181;L pipette. 
		CAUTION: Slices should not dry out, so remove and add fluids well by well.</li>
	</ol>
	</li>
	<li>Remove the PB with a plastic pipette and add 250 &#181;L of blocking buffer per well with a 1000 &#181;L pipette. Incubate for 3 h at room temperature (RT) on the shaker.</li>
	<li>During the incubation time, dilute the primary antibodies in antibody buffer in a reaction tube. Use 250 &#181;L antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 &#181;L pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing. 
	NOTE: To determine the background fluorescence, staining should also be performed without adding the primary antibody. For that, incubate the slice in antibody solution without primary antibodies according to the protocol.</li>
	<li>After the incubation time, remove the blocking buffer with a plastic pipette and add 250 &#181;L of antibody solution containing primary antibodies per well. Incubate slices with primary antibody overnight at 4 &#176;C on a shaker.</li>
	<li>Next day, wash the slices with washing buffer 1 3x for 10 min at RT on a shaker.
	<ol>
		<li>Remove the medium with a plastic pipette and add 300 &#181;L of washing buffer 1 per well. Incubate at RT for 10 min. Repeat 3 times.</li>
	</ol>
	</li>
	<li>During the washing steps, dilute the fluorophore-coupled secondary antibodies in antibody buffer in a reaction tube. Use 250 &#181;L antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 &#181;L pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing. 
	CAUTION: Because the antibodies are light-sensitive, all steps from this point on need to be performed in the dark.</li>
	<li>After the washing steps, remove the washing buffer with a plastic pipette and add 250 &#181;L of antibody solution containing secondary antibodies per well. Incubate the slices with secondary antibody for 90 min at RT in the dark.</li>
	<li>Wash the slices with washing buffer 2 3x for 10 min at RT.</li>
	<li>During the washing steps, dilute 4&#8242;,6-diamidino-2-phenylindole (DAPI) in 0.1 M PB in a concentration of 1:2000.</li>
	<li>Remove the washing buffer 2 with a plastic pipette and add 250 &#181;L of DAPI solution per well. Incubate for 5 min at RT on the shaker.</li>
	<li>Remove the DAPI solution with a plastic pipette and add 500 &#181;L of 0.1 M PB per well with a 1000 &#181;L pipette.</li>
	<li>Mount slices on microscope slides.
	<ol>
		<li>Place a microscope slide under the stereoscope. With a fine brush, add three separate drops of 0.1 M PB onto the slide. Place one slice per drop onto the microscope slide.</li>
		<li>Use the fine brush to flatten and orient the slices on the microscope slide.</li>
		<li>When all slices are positioned correctly, remove excess PB with a tissue and dry the slide carefully. 
		CAUTION: Avoid drying the brain slices completely.</li>
		<li>Add 80 &#181;L of embedding medium onto the slide. Carefully lower the coverslip onto the slide, thereby embedding the brain slices.</li>
		<li>Leave the slides to dry in the fume hood for 1-2 h (cover them to avoid light exposure) and store them in a microscope slide box at 4 &#176;C. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
2. Imaging
<ol>
	<li>After the embedding medium is completely hardened, place the microscope slide under the confocal microscope. 
	NOTE: Epifluorescence microscopy combined with deconvolution software should yield similar image quality.</li>
	<li>Adjust the laser settings by increasing or decreasing the laser intensity for every channel so that few pixels are overexposed to ensure maximum distribution of grey values.</li>
	<li>Acquire virtual tissues of the whole brain slice for the different channels.
	<ol>
		<li>In the imaging software, select the Tiles option and manually delineate the brain slice with the Tile Region Setup.</li>
		<li>Distribute support points throughout the tile region and adjust the focus for the different support points by pressing Verify Tile Regions/Positions&#8230;.</li>
		<li>Adjust the settings in Acquisition Mode according to the desired resolution and file size of the resulting image and start the scan.</li>
	</ol>
	</li>
	<li>When the scan is finished, use the Stitching function to process the virtual tissue. Export the file as a .tif with the function Image Export.</li>
</ol>
3. Computer-based Analysis
<ol>
	<li>Load all single channels for one image into FIJI by clicking File| Open.</li>
	<li>With the Freehand selection tool, delineate one hemisphere in the DAPI-channel. Create a mask of the selection by clicking Edit| Selection| Create mask.</li>
	<li>Determine the mean fluorescence intensity for the single channels (Mover and synaptophysin) by clicking Analyze| Measure. 
	NOTE: Make sure to select the different channels to determine the mean fluorescence intensity values for each channel.</li>
	<li>Copy the mean fluorescence intensity for the single channels into a spreadsheet.</li>
	<li>Determine the mean fluorescence intensity for the single channels in an area of interest by delineating the area also with the Freehand selection tool. Use a mouse brain atlas as reference.</li>
	<li>Repeat steps 3.1-3.5 for all hemispheres and all areas of interest. 
	NOTE: Determine the values for each hemisphere separately in order to later compare the values in an area of interest to that in the hemisphere (see step 4.2).</li>
</ol>
4. Data Handling
<ol>
	<li>In case the background fluorescence is high, a background subtraction might be needed. For that, determine the mean fluorescence intensity for the slice processed without primary antibody against the reference protein (here: synaptophysin) and subtract that value from all values obtained for the brain regions and hemispheres.</li>
	<li>When the mean fluorescence intensities for the single channels for every hemisphere and every area of interest have been determined (see Table 3), calculate the ratio of Mover to synaptophysin by dividing the value for Mover by the value for synaptophysin (yellow in Table 3). Perform this action for every hemisphere and every area of interest separately.</li>
	<li>Divide the ratio obtained for one area of interest by the ratio obtained for the corresponding hemisphere (orange in Table 3) to determine the ratio of the area of interest to the hemisphere.</li>
	<li>To determine the relative Mover abundance, translate the ratio obtained in 4.2 into a percentage by determining its deviation from 1 (red in Table 3). A ratio of 1.25 would therefore give a relative Mover abundance of 25% above average, and a ratio of 0.75 would yield a relative Mover abundance of 25% below average.</li>
</ol>
Table 1: Solutions used in this protocol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Fixative (500mL)</td>
		</tr>
		<tr>
			<td colspan="2">Mix 20g paraformaldehyde (total conc.: 4%) 
			50 mL 10x PBS stock solution (total conc.: 1x) 
			450 mL bidest H2OAdjust pH to 7.4 with NaOH 
			Note: To solve the paraformaldehyde in PBS, heat the solution. Do not heat over 70 oC, as PFA disintegrates at temperatures higher than 70 oC. 
			Caution: PFA is toxic, potentially carcinogenic and teratogenic. Wear gloves when working with PFA and work under the fume hood. Avoid ingestion.</td>
		</tr>
		<tr>
			<td colspan="2">0.1M PB (1L)</td>
		</tr>
		<tr>
			<td>Stock solution X 
			35.61 g Na2HPO4.2H2O in 1 L bidest H2O</td>
			<td>Stock solution Y 
			27.60 g NaH2PO4.2H2O in 1 L bidest H2O</td>
		</tr>
		<tr>
			<td colspan="2">Mix 385 mL stock solution X 
			115 mL stock solution Y 
			500 mL bidest H2O</td>
		</tr>
		<tr>
			<td colspan="2">Blocking buffer (50 mL)</td>
		</tr>
		<tr>
			<td colspan="2">Mix 1.25 mL normal goat serum (total conc.: 2.5%) 
			1.25 mL normal donkey serum (total conc.: 2.5%) 
			0.5 mL non ionic surfactant (Triton-X100, total conc.: 1%) 
			47 mL 0.1 M PB</td>
		</tr>
		<tr>
			<td colspan="2">Antibody buffer (50 mL)</td>
		</tr>
		<tr>
			<td colspan="2">Mix 0.25 mL normal goat serum (total conc.: 0.5%) 
			0.25 mL normal donkey serum (total conc.: 0.5%) 
			0.1 mL non ionic surfactant (total conc.: 0.2%) 
			49.4 mL 0.1 M PB</td>
		</tr>
		<tr>
			<td colspan="2">Washing buffer 1 (50 mL) </td>
		</tr>
		<tr>
			<td colspan="2">Mix 1 mL normal goat serum (total conc.: 2 %) 
			49 mL 0.1 M PB</td>
		</tr>
		<tr>
			<td colspan="2">Washing buffer 2 (50 mL) </td>
		</tr>
		<tr>
			<td colspan="2">Mix 0.5 mL normal goat serum (total conc.: 1 %) 
			49.5 mL 0.1 M PB</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies used in this protocol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Primary antibodies</td>
		</tr>
		<tr>
			<td>Directed against</td>
			<td>Host species</td>
			<td>RRID</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Mover</td>
			<td>Rabbit</td>
			<td>AB_10804285</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Synaptophysin</td>
			<td>Guinea pig</td>
			<td>AB_1210382</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td colspan="4">Secondary antibodies</td>
		</tr>
		<tr>
			<td>Target species</td>
			<td>Host species</td>
			<td>Fluorophore</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Rabbit</td>
			<td>Donkey</td>
			<td>AlexaFluor 647</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Guinea pig</td>
			<td>Goat</td>
			<td>AlexaFluor 488</td>
			<td>1:1000</td>
		</tr>
	</tbody>
</table>
Table 3: Example of data handling.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="6">Hemisphere</td>
		</tr>
		<tr>
			<td>Hemisphere #</td>
			<td>Mean fluorescence intensity Synaptophysin (A.U.)</td>
			<td>Mean fluorescence intensity Mover (A.U.)</td>
			<td>Ratio Mover/Synaptophysin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 
			2 
			3 
			4 
			5 
			6</td>
			<td>29.134 
			31.008 
			38.641 
			30.775 
			21.658 
			27.277</td>
			<td>22.810 
			24.046 
			29.324 
			25.444 
			18.091 
			23.364</td>
			<td>=C3/B3&#160; &#160; &#160; 0.783 
			=C4/B4&#160; &#160; &#160; 0.775 
			=C5/B5&#160; &#160; &#160; 0.759 
			=C6/B6&#160; &#160; &#160; 0.827 
			=C7/B7&#160; &#160; &#160; 0.835 
			=C8/B8&#160; &#160; &#160; 0.857</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="6">Hippocampus</td>
		</tr>
		<tr>
			<td>Hemisphere #</td>
			<td>Mean fluorescence intensity Synaptophysin (A.U.)</td>
			<td>Mean fluorescence intensity Mover (A.U.)</td>
			<td>Ratio Mover/Synaptophysin</td>
			<td>Ratio to hemisphere</td>
			<td>Relative Mover abundance (%)</td>
		</tr>
		<tr>
			<td>1 
			2 
			3 
			4 
			5 
			6</td>
			<td>35.26 
			33.955 
			41.231 
			39.853 
			30.129 
			28.737</td>
			<td>29.889 
			27.825 
			31.978 
			31.787 
			27.817 
			25.861</td>
			<td>=C11/B11&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.848 
			=C12/B12&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.819 
			=C13/B13&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.776 
			=C14/B14&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.798 
			=C15/B15&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.923 
			=C16/B16&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.900</td>
			<td>=D11/D3&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1.083 
			=D12/D4&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1.057 
			=D13/D5&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1.022 
			=D14/D6&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 0.965 
			=D15/D7&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1.105 
			=D16/D8&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1.051</td>
			<td>=(E11-1)*100&#160; &#160; &#160; 8.269&#160; 
			=(E12-1)*100&#160; &#160; &#160; 5.673 
			=(E13-1)*100&#160; &#160; &#160; 2.200 
			=(E14-1)*100&#160; &#160; &#160;-3.528 
			=(E15-1)*100&#160; &#160; 10.530 
			=(E16-1)*100&#160; &#160; &#160; 5.064</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL reaction tubes</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td>087721H</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic pipette (disposable)</td>
			<td>Sarstedt</td>
			<td>8,61,176</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL pipette</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml pipette</td>
			<td>Eppendorf</td>
			<td>3123000012</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Menzel microscope slides</td>
			<td>Fisher Scientific</td>
			<td>10144633CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM800</td>
			<td>Zeiss</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek</td>
			<td>Sakura</td>
			<td>4583</td>
			<td>OCT</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Merck</td>
			<td>1,06,34,60,500</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Merck Millipore</td>
			<td>S26-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>normal donkey serum</td>
			<td>Merck</td>
			<td>S30-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1,08,60,31,000</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-Mover</td>
			<td>Synaptic Systems</td>
			<td>RRID: AB_10804285</td>
			<td></td>
		</tr>
		<tr>
			<td>guinea pig anti-Synaptophysin</td>
			<td>Synaptic Systems</td>
			<td>RRID: AB_1210382</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-rabbit AF647</td>
			<td>Jackson ImmunoResearch</td>
			<td>RRID: AB_2492288</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse AF488</td>
			<td>Jackson ImmunoResearch</td>
			<td>RRID: AB_2337438</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN2 blue software</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscopy software</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>ImageJ</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying the distribution of a presynaptic protein in the mouse brain using immunofluorescence

Source: Wallrafen, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58940">Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence.</a> J. Vis. Exp. (2019).
This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain regions using confocal microscopy.

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunofluorescence

	...

Take a chemically fixed mouse brain slice embedded in a tissue-embedding medium.
Wash the slice with buffer to remove the medium.
Add a solution that permeabilizes the membranes and blocks non-specific binding sites.
Remove the solution.
To visualize neuronal synaptic proteins, incubate the slice with primary antibodies.
These antibodies target a presynaptic protein expressed in the synapses of specific brain regions and a ubiquitously expressed synaptic protein as a reference marker.
Wash with buffer to remove unbound antibodies.
Introduce fluorophore-labeled secondary antibodies that target the primary antibodies.
Wash with buffer to remove unbound secondary antibodies.
Counterstain the nuclei with a DNA-binding dye.
Wash with buffer and mount the slice using a suitable mounting medium.
Visualize the slice under a confocal microscope.
Calculate the mean fluorescence intensity ratios of the target protein and the reference marker to analyze the target protein&#39;s distribution across different brain regions.

quantifying-distribution-presynaptic-protein-mouse-brain-using

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Trauma

64 Views. Fonte: Wallrafen, R., et al. Quantificazione della distribuzione eterogenea di una proteina sinaptica nel cervello di topo utilizzando l'immunofluorescenza. J. Vis. Exp. (2019). Questo video dimostra la colorazione in immunofluorescenza di una fetta di cervello di topo per quantificare la distribuzione di una specifica proteina presinaptica. Il processo prevede il blocco di siti aspecifici, l'applicazione di anticorpi primari per la proteina bersaglio e un marcatore di riferimento, seguiti dall...

Video: Quantificazione della distribuzione di una proteina presinaptica nel cervello di topo mediante immunofluorescenza - Concept

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

Preparazione e Immunostaining di su colture organotipiche Myelinating fetta cerebellare

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

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JoVE Journal

Neuroscienze

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

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Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence