Name: Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain (Video) | JoVE Journal
Uploaded: 2019-05-23T15:00:00.000+00:00
Duration: 6 min 4 s
Description: Learn near-infrared fluorescence and high-resolution scanning to measure protein expression in rat brain regions. This immunohistochemistry protocol enables quantification of pan and phosphoproteins for studying molecular signaling pathways underlying cognition.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 anterior and clear from top of skull.</li>
	<li>Use rongeurs to carefully remove the back part of skull, and then using small dissecting scissors cut down midline of skull, pushing upward against the top of skull at all times to avoid damaging the brain tissue.</li>
	<li>Use rongeurs to peel away right and left half of skull to expose the brain.</li>
	<li>Use a small spatula to scoop under brain and sever nerves, carefully elevate brain and freeze brains in isopentane chilled on dry ice (at least -20 &#176;C). After this, store brains in a -80 &#176;C freezer until slicing.</li>
	<li>Slice brains in regions of interest at 30&#8211;50 &#181;m in a cryostat maintained between -9 &#176;C and -12 &#176;C. Directly mount slices onto glass slides and store in a -80 &#176;C freezer until time of immunocytochemistry assay. 
	NOTE: In this protocol brain regions of interest were the hippocampus and the amygdala.</li>
</ol>
2. Single Immunohistochemical Reaction
NOTE: For double immunohistochemical reaction, the protocol is the same as the single immunohistochemical reaction except this reaction has two primary antibodies of different hosts (e.g., rabbit and mouse) and two secondary antibodies for the corresponding primaries should be from a single host (e.g., goat antirabbit and goat antimouse). The secondary antibodies also have to be from two different spectra that are available in high-resolution scanners. For example, one secondary antibody with an emission spectrum peak at 680 nm and one secondary antibody 800CW (emission spectrum peak at 780 nm).
<ol>
	<li>Remove glass slides from the freezer and allow to equilibrate to room temperature for 30 min.</li>
	<li>Under a fume hood, fix brain tissue in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 1&#8722;2 h at room temperature.</li>
	<li>Rinse slides in 0.1 M tris buffered saline (TBS) three times for 10 min each. Permeabilize cell membranes by incubating slides in a mild detergent (e.g., 0.01% detergent) for 30&#8722;60 min.</li>
	<li>Rinse slides in TBS three times for 15 min each.</li>
	<li>Dilute primary antibody for protein of interest in PBS in the correct concentration. For example, to detect the immediate early gene c-Fos, prepare a rabbit primary anti c-Fos antibody in a concentration of 1:500.</li>
	<li>Pipette primary antibody solution directly onto the brain tissue (approximately 200 &#181;L per 3 inch x 1 inch slide).</li>
	<li>Use coverslips to incubate the brain tissue on glass slides in primary antibody dilution for either 1&#8722;2 h at room temperature or overnight (~17 h) at 4 &#176;C.</li>
	<li>Remove coverslips and wash slides in TBS that has a small amount of detergent added to it (e.g., 0.01% detergent, &#34;TBS-T&#34;) four times for 15 min each.</li>
	<li>Use coverslips to incubate brain sections in a secondary antibody at room temperature for 2 h. 
	NOTE: The secondary antibody has to be to the correct dilution and in a diluent containing TBS, detergent, and 1.5% of host serum. For example, a goat secondary antibody in a dilution of 1:2,000 would contain 1.5% goat serum and 0.05% detergent.</li>
	<li>Rinse slides in TBS-T four times for 20 min each, and then in TBS four times for 20 min each.</li>
	<li>Dry slides at room temperature in the dark overnight. When slides are dry, they are ready for imaging.</li>
</ol>
3. Imaging
<ol>
	<li>Place slides onto the near-infrared scanning interface with the tissue facing down. Either image one glass slide or multiple slides at a time using a selection tool.</li>
	<li>Image slides using the highest quality setting with an offset of 0 nm and a resolution of 21 &#181;m. The scanning will typically take 13&#8722;19 h depending on the scanning equipment used.</li>
	<li>Import images into the image analysis software (e.g., ImageStudio) to view and mark for semi-quantitative protein analysis.</li>
</ol>
4. Protein Expression Analysis
<ol>
	<li>Open the image analysis software and select the Work Area into which the image was scanned.</li>
	<li>Open the scanned image in the image analysis software to view the scan, and adjust which wavelengths are viewed, as well as the contrast, brightness, and magnification shown without altering the raw image or the total quantified emission.</li>
	<li>Identify the key regions for quantification and select the Analysis tab along the top of the page, then select Draw Rectangle (or Draw Ellipse/ Draw Freehand) to draw a rectangle over the area that will be quantified.</li>
	<li>To view the size of the rectangle, select Shapes along the bottom left of the screen, then select Columns along bottom right. Add Height and Width columns to identify the shape size. 
	NOTE: It is important to control for shape size when comparing quantification. It is recommended to use identical shapes placed within the desired quantification location, in order to get an accurate sampling of the emissions for that region.</li>
	<li>Then name the shape and repeat. Once all regions are sampled the data available from the columns tab can be aggregated and analyzed.</li>
</ol>

<ol><li>Kandel, E. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKandel%2C+ER+%22Principles+of+Neural+Science%22">Principles of Neural Science</a>. , McGraw Hill. New York. (2013).</li>
<li>Byrne, J. H., Roberts, J. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aByrne%2C+JH+%22From+Molecules+to+Networks%3A+An+Introduction+to+Cellular+and+Molecular+Neuroscience%22">From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience</a>. , Academic Press/Elsevier. (2009).</li>
<li>Salami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+D2%2F3+binding+potential+modulates+neural+signatures+of+working+memory+in+a+load-dependent+fashion%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Dopamine D2/3 binding potential modulates neural signatures of working memory in a load-dependent fashion.</a> Journal of Neuroscience. , (2018).</li>
<li>Merali, Z., Khan, S., Michaud, D. S., Shippy, S. A., Anisman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+amygdaloid+corticotropin-releasing+hormone+%28CRH%29+mediate+anxiety-like+behaviors%3F+Dissociation+of+anxiogenic+effects+and+CRH+release%5BTitle%5D)+AND+%22European+Journal+of+Neuroscience%22%5BJournal%5D)">Does amygdaloid corticotropin-releasing hormone (CRH) mediate anxiety-like behaviors? Dissociation of anxiogenic effects and CRH release.</a> European Journal of Neuroscience. 20 (1), 229-239 (2004).</li>
<li>English, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dataset+of+mouse+hippocampus+profiled+by+LC-MS%2FMS+for+label-free+quantitation%5BTitle%5D)+AND+%22Data+in+Brief%22%5BJournal%5D)">Dataset of mouse hippocampus profiled by LC-MS/MS for label-free quantitation.</a> Data in Brief. 7, 341-343 (2016).</li>
<li>David, M. A., Tayebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+protein+aggregates+in+brain+and+cerebrospinal+fluid+derived+from+multiple+sclerosis+patients%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">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/pubmed?term=((Immunocytochemical+methods+and+protocols%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Immunocytochemical methods and protocols.</a> Methods in Molecular Biology. , (2010).</li>
<li>Spitzer, N., Sammons, G. S., Price, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autofluorescent+cells+in+rat+brain+can+be+convincing+impostors+in+green+fluorescent+reporter+studies%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies.</a> Journal of Neuroscience Methods. 197 (1), 48-55 (2011).</li>
<li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+c-Jun+to+identify+fear+extinction+learning-specific+patterns+of+neural+activity+that+are+affected+by+single+prolonged+stress%5BTitle%5D)+AND+%22Behavioural+Brain+Researach%22%5BJournal%5D)">Using c-Jun to identify fear extinction learning-specific patterns of neural activity that are affected by single prolonged stress.</a> Behavioural Brain Researach. 341, 189-197 (2018).</li>
<li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+circuits+via+which+single+prolonged+stress+exposure+leads+to+fear+extinction+retention+deficits%5BTitle%5D)+AND+%22Learning+%26+Memory%22%5BJournal%5D)">Neural circuits via which single prolonged stress exposure leads to fear extinction retention deficits.</a> Learning & Memory. 23 (12), 689-698 (2016).</li>
<li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPA+receptor+trafficking+and+synaptic+plasticity%5BTitle%5D)+AND+%22Annual+Review+of+Neuroscience%22%5BJournal%5D)">AMPA receptor trafficking and synaptic plasticity.</a> Annual Review of Neuroscience. 25, 103-126 (2002).</li>
<li>Nabavi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+a+memory+with+LTD+and+LTP%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Engineering a memory with LTD and LTP.</a> Nature. 511 (7509), 348-352 (2014).</li>
<li>Kimmelmann-Shultz, B., Mohammadmirzaei, N., Della Valle, R., Knox, D. Using high resolution near infrared imaging to measure fear-learning induced changes in AMPA/NMDA ratios throughout the fear circuit. The Annual Delaware Neuroscience Research and Poster Symposium, Newark, DE, , (2018).</li>
<li>Eagle, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+prolonged+stress+enhances+hippocampal+glucocorticoid+receptor+and+phosphorylated+protein+kinase+B+levels%5BTitle%5D)+AND+%22Neuroscience+Research%22%5BJournal%5D)">Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels.</a> Neuroscience Research. 75 (2), 130-137 (2013).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Brain Extraction</td><td></td><td></td><td></td></tr><tr><td>Anesthesia Induction Chamber</td><td>Kent Scientific</td><td>VetFlo-0530SM</td><td></td></tr><tr><td>Kleine Guillotine</td><td>Harvard Apparatus</td><td>73-1920</td><td></td></tr><tr><td>Friedman Rongeur</td><td>Fine Science Tools</td><td>16000-14</td><td>used to remove back of skull</td></tr><tr><td>Delicate Dissecting Scissors</td><td>Fischer Scientific</td><td>08-951-5</td><td>used to cut upward along midline of skull</td></tr><tr><td>Micro Spatula</td><td>Fischer Scientific</td><td>21-401-5</td><td>used to scoop out brain</td></tr><tr><td>Glass Microscope Slides</td><td>Fischer Scientific</td><td>12-549-6</td><td></td></tr><tr><td>Immunohistochemical Reaction</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Triton X-100</td><td></td><td></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&amp;nbsp;</td></tr><tr><td>Tween-20</td><td></td><td></td><td>Used as a small amount of detergent added to TBS&amp;nbsp; to procuce TBS-T after coverslipping slides with primary antibody</td></tr><tr><td>Licor Odyssey scanner</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr><tr><td>Image Studio</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr></tbody></table>

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

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...

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 ...

using-near-infrared-fluorescence-high-resolution-scanning-to-measure

Research

JoVE Journal

Neuroscience

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices.  By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

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...

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

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal...

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

Source: Kimmelmann-Shultz, B., et. al. Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain. J. Vis. Exp. (2019) This video demonstrates a near-infrared fluorescence and high-resolution scanning method for measuring protein expression in the rodent brain.

Measuring Protein Expression in the Rodent Brain Using Near-Infrared Fluorescence and High-Resolution Scanning 

Source: Lacin, E., et al. Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo. J. Vis. Exp. (2016)This video demonstrates a technique for imaging neurotransmitter release in vivo using cell-based neurotransmitter fluorescent engineered reporters (CNiFERs), specialized reporter cells pre-injected into the mouse cortex. Positioned under a two-photon microscope, the mouse allows visualization of...

Imaging Neurotransmitter Release Using Cell-Based Neurotransmitter Fluorescent Engineered Reporters

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

Protein synthesis is a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

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.

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

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Learn near-infrared fluorescence and high-resolution scanning to measure protein expression in rat brain regions. This immunohistochemistry protocol enables quantification of pan and phosphoproteins for studying molecular signaling pathways underlying cognition.

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

Explore More Videos