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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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 ...

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