Name: using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain
Uploaded: 2019-05-23T15:00:00
Description: Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

The research in this report was funded by a target grant from the NIGMS (1P20GM103653) awarded to DK.

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware. Male Sprague Dawley rats approximately 55&#8211;75 days old were used for this protocol.
1. Brain Extraction and Tissue Preparation
<ol>
	<li>Anesthetize rat with isoflurane in anesthesia induction chamber until rat no longer exhibits a response to foot pinch.</li>
	<li>Sacrifice rats via rapid decapitation using a guillotine.</li>
	<li>Cut skin on skull posterior to ...

<ol>
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A., Tayebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein+aggregates+in+brain+and+cerebrospinal+fluid+derived+from+multiple+sclerosis+patients.">Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.</a> Frontiers in Neurology. 5, 251 (2014).</li><li>Oliver, C., Jamur, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+methods+and+protocols.">Immunocytochemical methods and protocols.</a> Methods in Molecular Biology. , (2010).</li><li>Spitzer, N., Sammons, G. S., Price, E. 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The results presented in this article show that near-infrared immunocytochemistry in combination with high-resolution scanning can be used to obtain semi-quantitative measures of protein expression in brain tissue. It can also be used to label two proteins simultaneously in the same brain region. We have previously used near-infrared immunohistochemistry to measure immediate early gene expression in multiple brain regions9,10. Immediate early genes can be used as...

The study of neuroscience concerns an investigation of how cells in the brain mediate specific functions1. These can be cellular in nature such as how glia cells confer immunity in the central nervous system or can involve experiments that aim to explain how the activity of neurons in the dorsal hippocampus leads to spatial navigation. In a broad sense, cellular function is determined by the proteins that are expressed in a cell and the activity of these proteins2. As a result, measuring the expression and/or activity of proteins in brain cells are critical for the study of neuroscience.
A number of techniques are available to measure protein expression in the brain. These include in vivo methods such as positron emission topography for receptor densities3 and micro-dialysis for small peptides4. More commonly, ex vivo methods are used to examine protein function and expression. These include mass spectrometry techniques5, western blot and enzyme-linked immunosorbent assay (ELISA)6, and immunocytochemistry7. Immunocytochemistry is widely used in the field of neuroscience. This technique involves the use of a primary antibody to detect a protein (or antigen) of interest (e.g., c-Fos) and a conjugated secondary antibody to detect the protein-primary antibody complex (Figure 1). To enable detection of the protein-primary antibody-secondary antibody complex, secondary antibodies have oxidizing agents such as horseradish peroxidase (HRP) conjugated to them. This allows for the formation of precipitates in cells that can be detected using light microscopy7. Secondary antibodies can also have chemicals fluoresce conjugated to them (i.e., fluorophores). When stimulated these chemicals emit light, which can be used to detect protein-primary antibody-secondary antibody complexes7. Lastly, sometimes primary antibodies have reducing agents and fluorescence chemicals attached to them directly negating the need for secondary antibodies7 (Figure 1).
Interestingly, many immunocytochemistry methods allow for visualization of proteins in brain cells, but not the ability to quantify the amount of protein in a specific cell or brain region. Using light microscopy to detect precipitates from reduction reactions allows for visualization of neurons and glia, but this method cannot be used to quantify protein expression in cells or in a specific brain region. In theory, fluorescence microscopy can be used for this, because the light emitted from the fluorescent secondary antibody is a measure of the protein-primary antibody-secondary antibody complex. However, autofluorescence in brain tissue can make it difficult to use fluorescence microscopy to quantify protein expression in brain tissue8. As a result, light emitted from fluorescent images of brain tissue is rarely used to quantify protein expression in the brain.
Many of these issues can be addressed using near-infrared immunocytochemistry in conjunction with high-resolution scanning9,10. In this article, we describe how immunocytochemistry coupled with fluorophores in the near-infrared emission spectra can be combined with high resolution scanning (e.g., 10&#8211;21 &#181;m) to obtain sharp images that allow for semi quantification of protein in distinct brain regions.

<table border="0" cellpadding="0" cellspacing="0" style="width:658px;" width="657">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:658px;">Brain Extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Anesthesia Induction Chamber</td>
			<td style="width:147px;">Kent Scientific</td>
			<td>VetFlo-0530SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kleine Guillotine</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td>73-1920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Friedman Rongeur</td>
			<td style="width:147px;">Fine Science Tools</td>
			<td>16000-14</td>
			<td>used to remove back of skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Delicate Dissecting Scissors</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td>08-951-5</td>
			<td>used to cut upward along midline of skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Micro Spatula</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td>21-401-5</td>
			<td>used to scoop out brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glass Microscope Slides</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td style="width:114px;">12-549-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:658px;">Immunohistochemical Reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;Triton X-100</td>
			<td style="width:147px;"></td>
			<td style="width:114px;"></td>
			<td>Used as a mild detergent to permeabilize cells after fixing in Paraformaldehyde, also used as mild detergent in combination with host serum and secondary antibody&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween-20</td>
			<td style="width:147px;"></td>
			<td style="width:114px;"></td>
			<td>Used as a small amount of detergent added to TBS&#160; to procuce TBS-T after coverslipping slides with primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Licor Odyssey scanner</td>
			<td style="width:147px;">Licor Biotechnology Inc.</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Image Studio</td>
			<td style="width:147px;">Licor Biotechnology Inc.</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Prior to using high-resolution scanning for immunohistochemistry, one should verify that the protocol works. This can be accomplished using a validation assay where brain sections from the same animal are incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody. Results for such a validation assay are shown in Figure 2. In this reaction we were detecting the immediate early gene c-Jun in the dorsal h...

Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

This protocol is significant because it allows for quantification of two proteins in the same brain region. This allows the experimenter to look at activity in molecular signaling pathways in brain regions by assaying pan and phosphoproteins. It can also be used to assay proteins that are markers of different cognitive phenomena.We are using a relatively simple technique, such as immunohistochemistry, to answer questions that typically require more involved techniques. Including examining the activity of molecular signaling pathways, examining AMPA/NMDA ratio. Using a cryostat maintained between minus 9 degrees Celsius and minus 12 degrees Celsius, slice previously isolated and frozen rat brain in regions of interest at 30-50 micrometers.Directly mount slices onto glass slides and store at minus 80 degrees Celsius until immunocytochemistry. Remove the glass slides from the freezer and allow them to equilibrate to room temperature for 30 minutes. Working under a fume hood, place the slides in 4%paraformaldehyde in 0.1 molar PBS for one to two hours at room temperature for tissue fixation.Then rinse the slides in 0.1 molar TBS three times for 10 minutes each. To permeabilize cell membranes, incubate the slides in a mild detergent for 30-60 minutes. And rinse again in TBS three times for 15 minutes each.Dilute primary antibody for protein of interest in PBS in the correct concentration as described in the manuscript. Pipette primary antibody solution directly onto the brain tissue. Place cover slips on the slides and incubate the brain tissue in primary antibody dilution for 1-2 hours at room temperature.After incubation, remove the cover slips and wash the slides in TBS that has a small amount of detergent added to it, four times for 15 minutes each. Dilute secondary antibody in a diluent containing TBS, detergent, and 1.5%of host serum. Add secondary antibody to the slides, cover with cover slips and incubate at room temperature for two hours.After incubation, rinse the slides in TBS-T four times for 20 minutes each and then in TBS four times for 20 minutes each. Dry the slides at room temperature in the dark, overnight. Place slides onto near-infrared scanning interface with the tissue facing down.Image multiple slides at a time using the selection tool. Image the slides using the highest quality setting with a resolution of 21 micrometers. In an offset of zero nanometers, import images into image analysis software to view and mark for semiquantitative protein analysis.After opening the image analysis software, select the work area into which the image was scanned. Then open the scanned image in the image analysis software to view the scan and adjust wavelengths, contrast, brightness, and magnification shown without altering the raw image or the total quantified emission. After identifying the key regions for quantification, select the analysis tab along the top of the page, then select draw rectangle to draw a rectangle over the area that will be quantified.To view the rectangle size, select shapes along the bottom left of the screen. Then select columns along the bottom right. Then add height and width columns to identify the shape size.Finally, name the shape and repeat. Once all regions are sampled, proceed with combining and analyzing of the data available from the columns tab. To verify that this protocol works, brain sections were incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody.The immediate early gene c-jun expression was detected in the dorsal hippocampus and amygdala only when both primary and secondary antibodies were applied to the brain tissue. GluR1 sub-unit of AMPA receptor and the NR2A sub-unit of NMDA receptor were assayed in the stool protein detection in amygdala nuclei. This allowed examining the ratio of AMPA NMDA receptors in sub-nuclei of the amygdala which is a neurobiological signature of learning and memory.Mean and normalized measures of protein expression were obtained from high resolution scans in the ventral hippocampus. Using the image analysis software, a rectangle is placed in the region of interest and mean intensity of light from the shape was used as a measure of fluorophore expression, which is a measure of protein expression. To obtain the normalized curve, a shape was placed across a region that expresses high signal and low signal in the area under the curve was used as a measure of protein expression.The biggest struggle with this procedure is finding an antibody and making sure that it works. The application is simple. My advice would be to first attempt a regular immunohistochemical procedure, then try this technique.Always performing a validation assay first. The most important thing to remember is to check your antibody dilution and make sure it's applied correctly. Without these steps, the procedure will fail.

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