We thank Irmgard Weiss for excellent technical assistance. The authors acknowledge support by Hermes Pofantis and Andoniya Petkova. The authors also thank the European Neuroscience Institute for the usage of the LSM800 and technical assistance, especially by Dr. Nils Halbsgut. This work was funded by the University Medical Center G&#246;ttingen. JSV acknowledges support by the Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).

This protocol does not involve experiments on live animals. Experiments involving euthanizing of animals to obtain brain samples were approved by the local animal protection authorities (Tierschutzkommission der Universit&#228;tsmedizin G&#246;ttingen) under the approval number T 10/30.
NOTE: For this protocol, 3 adult male C57BL/6 mice were used.
1. Sample Preparation
<ol>
	<li>Prepare fixative and 0.1 M phosphate buffer (PB; see Table 1).</li>
	<li>Fix the animal by transcardial perfusion as described in Gage et al.28. First wash out the blood with 0.9% NaCl-solution, then perfuse with 30 mL of 4% paraformaldehyde (PFA).</li>
	<li>Open the skull with scissors and carefully isolate the brain using a spoon with blunt edges to avoid damaging the tissue.</li>
	<li>Fill a 50 mL reaction tube with fixative and postfix the brain in 4% PFA at 4 &#176;C overnight.</li>
	<li>Remove the fixative and wash the brain in 50 mL of 0.1 M PB on a shaker for 30 min.</li>
	<li>After washing, incubate the brain in a 50 mL reaction tube in 30% sucrose in 0.1 M PB for 48 h or until it sinks in the tube at 4 &#176;C for cryoprotection.</li>
	<li>Trim the cryoprotected brain with a sharp blade, place it in a cryomold, and embed it with optimal cutting temperature (OCT) compound. Avoid bubbles. Orient the brain and freeze the cryomold in the -80 &#176;C freezer.</li>
	<li>Mount the frozen tissue for sectioning. Equilibrate the tissue to the cryomicrotome temperature for at least 15 min before sectioning.</li>
	<li>Section the brain into 25 &#181;m thick coronal slices. Touch the OCT carefully with a glass hook without touching the brain tissue. Collect 3 adjacent slices per well in a 24 well plate and store them in 0.1 M PB at 4 &#176;C until staining. 
	NOTE: The protocol can be paused here for up to two weeks. Longer storage times can interfere with the tissue quality and thus influence the outcome of the experiment.</li>
</ol>
2. 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 PB to remove excess OCT.
	<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, stainings 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>
3. 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 (see Table of Materials), 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>
4. Computer-based Analysis
<ol>
	<li>Load all single channels for one image into FIJI29 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 4.1-4.5 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 5.2).</li>
</ol>
5. Data Handling
<ol>
	<li>In case the background fluorescence is high (see Discussion), 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 5.2 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>

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The authors have nothing to disclose.

The method presented here aims at quantifying the distribution of a protein of interest relative to the abundance of a marker protein with a known distribution. Immunofluorescence staining can show a high variability of staining intensities between different slices. The quantification approach described here circumvents this problem by determining the ratio of the protein of interest to the average across the hemisphere. Therefore, different staining intensities across slices are cancelled out and allow for a quantitativ...

Communication between neurons happens at specialized contact sites called synapses. Synapses contain a myriad of different proteins that orchestrate synaptic transmission. Some of those proteins show a heterogeneous distribution throughout the nervous system and are not present in every synapse1. One example for such a protein is Munc13, which is involved in the priming process of synaptic vesicles. There are different isoforms of Munc13, which are heterogeneously distributed throughout the brain2, and the presence or absence of specific isoforms can influence short-term synaptic plasticity and synaptic vesicle dynamics3,4,5. Therefore, it is of vital importance to be able to identify the presence of different synaptic proteins across brain areas.
The methods of choice for quantification of synaptic proteins - so far - are mass spectrometry and Western blotting, rather than immunohistochemistry6,7,8,9. In some cases, several methods are used to complement each other to assess both the quantity and the localization of specific proteins (i.e., Wilhelm et al.10). The method we describe here allows for the localization and quantification of proteins of interest without the need of using any biochemical method, simply employing immunofluorescent stainings. Another advantage here is that the quantification can be done over areas much smaller and, therefore, more specific, than those achieved by other methods. However, one has to take into consideration that a reliable reference protein is needed to assess the distribution of the protein of interest.
Fluorescent staining by immunohistochemistry allows us to routinely identify the localization of proteins across brain areas as well as within different neuronal compartments. To identify the different compartments, specific markers are used. Typically, antibodies against synapsin and synaptophysin11 can be used to label synaptic vesicles, while antibodies against bassoon label the active zone of a presynaptic terminal12. Vesicular transporters, such as the vesicular glutamate transporters (vGluT) or vesicular GABA transporter (vGAT), are used to label excitatory13 and inhibitory14 presynaptic terminals, respectively. On the postsynaptic side, antibodies against the Homer protein can be employed to mark postsynaptic terminals, and antibodies against postsynaptic density protein 95 (PSD95)15,16,17 or gephyrin18,19,20 can label excitatory or inhibitory postsynaptic terminals, respectively. By using antibodies against a protein of interest and markers such as the ones described above, one can determine the localization of such protein. Many studies to date have done this in a qualitative manner21. However, to reliably determine the differential distribution of a specific synaptic protein, one must not only determine its presence or absence but also its relative concentration. The heterogeneity of sizes and density of synapses makes it important to establish a ratio between the synaptic marker and the protein of interest. Otherwise, synapse-rich regions such as the non-pyramidal layers of the hippocampus and the molecular layer of the cerebellum will show a high density of synaptic proteins, only due to the higher density of synapses but not due to a strong presence of that protein in each synapse (e.g., Wallrafen and Dresbach1). On the other hand, proteins in the neuronal soma (e.g., TGN3822) will usually show strong presence in the hippocampal pyramidal cell layer or hippocampal or cerebellar granule cell layer due to the high concentration of neuronal cell bodies in those areas. Therefore, this non-homogeneous distribution of structures, in this case synapses, can lead to a false estimation of the distribution of the protein of interest itself. Furthermore, there is an intrinsic variability in staining intensities across samples in immunohistochemical stainings. The protocol described here takes this into consideration and avoids such biases, as well as other caveats that arise from immunohistochemical methods.
In our recent study, we have used this method to describe the differential expression of Mover (also called TPRGL23 or SVAP3024) across 16 different brain areas1. Mover is a vertebrate-specific synaptic protein that can be found in association to synaptic vesicles and influences neurotransmitter release25,26,27. We have related the Mover expression to the abundance of synaptic vesicles, by staining for synaptophysin as a synaptic vesicle reference marker. We found high levels of Mover particularly in the septal nuclei, the ventral pallidum, and the amygdala. Within the hippocampus, we found a heterogeneous distribution of Mover, with high levels in the layers associated with intra-hippocampal computation, and low levels in input- and output layers.

<table><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>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 087721H</td><td></td></tr><tr><td>plastic pipette (disposable)</td><td>Sarstedt</td><td>861,176</td><td></td></tr><tr><td>1000 mL pipette</td><td>Rainin</td><td>&amp;nbsp;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&amp;nbsp;</td><td>IKA</td><td>3340001</td><td></td></tr><tr><td>Menzel microscope slides</td><td>Fisher Scientific</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 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>freezing microtome</td><td>Leica</td><td></td><td></td></tr><tr><td>PBS (10X)</td><td>Roche</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 11666789001</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; P6148-1kg</td><td></td></tr><tr><td>NaCl</td><td>BioFroxx</td><td>1394KG001</td><td></td></tr><tr><td>sucrose</td><td>neoFroxx</td><td>1104KG001</td><td></td></tr><tr><td>Tissue Tek</td><td>Sakura&amp;nbsp;</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>1,063,460,500</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>1,086,031,000</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>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 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 heterogeneous distribution of a synaptic protein in the mouse brain using immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Representative staining patterns of different markers can be seen in Figure 1. The pattern varies depending on the distribution of the protein. Examples of five rostro-caudal levels are shown in columns (A)-(E). A representative DAPI staining is shown in the first row: DAPI adheres to the DNA of a cell and thus nuclei are stained. This results in a punctate pattern. Regions with a high cell density are brighter than regions w...

Watch this Scientific Journal Video about Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence at JoVE.com

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

quantifying-heterogeneous-distribution-synaptic-protein-mouse-brain

Research

JoVE Journal

Neuroscience

7.9K Views.  University Medical Center Göttingen. Hier beschrijven we een protocol met immunofluorescentie, confocale microscopie en computergebaseerde analyse om de verdeling van synaptisch eiwit ten opzichte van een referentie-eiwit te bepalen. Onze methode omzeilt de inherente variabiliteit van immunofluorescentie vlekken met behulp van een verhouding in plaats van absolute fluorescentie niveaus. Daarnaast u met deze methode de heterogeniteit van eiwitdistributie op verschillende niveaus analyseren.Van hele hersenschijfjes tot hers...

Video: Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological ...

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

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

	...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

<a href='https://app.jove.com/v/56153/evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices'>Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).</a>This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic architecture through their colocalization.

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for ...

Neuroimaging

Synaptic & Cellular Imaging

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

<a href='https://app.jove.com/v/53853/combined-optogenetic-freeze-fracture-replica-immunolabeling-to'>Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)</a>This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

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