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

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

To prepare the slices for immunostaining, use a plastic pipette to remove the PB solution from one well without drawing in the brain slices. Then, use a 1,000-microliter pipette to add 250 microliters of fresh PB to wash them of excess OCT. Repeat this washing for each well at the time to avoid drying out the slices.
Then, use a plastic pipette to remove the PB solution from the first well. Use a 1,000-microliter pipette to add 250 microliters of blocking buffer per well, working well by well again. Incubate the plate at room temperature on a shaker for three hours. During the incubation at 250 microliters of antibody buffer per well to a reaction tube.
Then, use a 2-microliter pipette to add the appropriate amount of antibody, pipetting it directly into the solution, and gently pipette up and down several times to mix. Then, vortex this diluted antibody to ensure proper mixing.
Working well by well, remove the blocking buffer with a plastic pipette and add 250 microliters of primary antibody solution per well. Incubate the plate on a shaker at 4 degrees Celsius overnight.
The following day, remove the antibody solution with a plastic pipette. Wash the slices with 300 microliters of washing buffer 1 per well, three times 10 minutes each wash on a shaker at room temperature. During the washing, working in the dark, dilute the fluorophore-coupled secondary antibody in a reaction tube in the same fashion as previously done with the primary antibody.
After completed washing, remove the washing buffer with a plastic pipette and add 250 microliters of secondary antibody solution per well. Incubate in the dark at room temperature for 90 minutes. After completed incubation, remove the antibody solution with a plastic pipette. Wash the sections three times with washing buffer 2 in the same way as with washing buffer 1.
During this washing, dilute DAPI stain in 0.1 molar PB to achieve a 1-to-2000 concentration. After removing the washing buffer from the plate, add 250 microliters of DAPI solution, and incubate at room temperature on the shaker for 5 minutes.
After removing the DAPI solution with a plastic pipette, use a 1,000-microliter pipette to add 500 microliters of 0.1 molar PB per well. Place a microscope slide under a stereoscope. Use a fine brush to add three separate drops of 0.1 molar PB onto the slide. Using the brush, place one slice per drop on the slide, and then, flatten and orient the slices.
After all slices are positioned correctly, use a paper tissue to remove excess PB and dry carefully without drying the slices completely. Then, add 80 microliters of embedding medium onto the slide, and carefully cover it with a coverslip to embed the brain slices.
Cover the slides to avoid light exposure and leave them to dry in the fume hood for one to two hours, and then store them in a microscope slide box at 4 degrees Celsius until ready for confocal microscopy.
After acquiring virtual tissues of the whole brain slice for different channels as described in the manuscript, load all single channels for one image into Fiji by clicking File and then Open. Then, use the freehand selection tool to delineate one hemisphere in the DAPI channel. Click on Edit, then Selection, and then create mask to create a mask of the selected region.
Then click on Analyze, and then Measure Particles to determine the mean fluorescence intensity for single channels, making sure to select different channels to determine the mean fluorescence intensity values for each channel.
After that, copy the mean fluorescence intensity for the single channels into a spreadsheet. To determine the mean fluorescence intensity for the single channels in an area of interest, delineate the area with the freehand selection tool.

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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>

Source: Wallrafen, R., et al. <a href="https://app.jove.com/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.

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.

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

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

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