Name: imaging neurons within thick brain sections using the golgi-cox method
Uploaded: 2017-04-18T13:00:00
Description: Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

This work was supported by a Discovery Grant to CDCB from the Natural Sciences and Engineering Research Council of Canada (NSERC), a John R. Evans Leaders Fund research infrastructure grant to CDCB from Canada Foundation for Innovation (CFI project number 30381), and by a Discovery Grant to NJM from NSERC. ELL was supported by an Ontario Graduate Scholarship and by an OVC Scholarship from the Ontario Veterinary College at the University of Guelph. CDS was supported by an Undergraduate Student Research Assistantship from NSERC. ALM was supported by an Alexander Graham Bell Scholarship from NSERC and by an OVC Scholarship from the Ontario Veterinary College at the University of Guelph.

Adult female CD1-strain mice were used in this study. Similar staining can be accomplished using both sexes at various ages. Experimental animals were cared for according to the principles and guidelines of the Canadian Council on Animal Care, and the experimental protocol was approved by the University of Guelph Animal Care Committee.
1. Golgi-Cox Staining
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	<li>Golgi-Cox Impregnation of Brains
	<ol>
		<li>Make the Golgi-Cox solution of 1% (w/v) potassium dichromate, 0....

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We describe here a Golgi-Cox staining protocol along with two useful variants for visualizing neurons within thick brain sections. As shown in the Representative Results, the use of a high-resolution objective that has a long 800 &#956;m working distance allows for the reliable visualization of entire neurons throughout the depth of brain sections cut at 500 &#956;m. This study of relatively thick brain sections increases the probability that stained neurons of any type are fully contained within the slice, which is espe...

The visualization of individual neurons within tissue samples allows for the in situ analysis of neuron morphological characteristics, which has significantly advanced our understanding of the brain and how it may be influenced by endogenous disease or exogenous environmental factors. The Golgi-Cox staining method is a cost-effective, relatively simple means of staining a random sample of neurons within the brain. First developed by Golgi1 and modified by Cox2 in the 1800s, researchers have further refined this technique over the years to produce clear, well-stained neurons that can be used to visualize and quantify both dendritic tree morphology and spine density3,4,5,6,7,8,9.
A major technical consideration for the visualization of stained neurons within brain sections is the maximum slice thickness, which is limited by the working distance of available high-magnification/high-resolution microscope objectives. Common oil-immersion objectives in the 60 - 100X range provide excellent resolution, but are limited by their working distances that are typically no greater than 200 &#956;m. Brain sections cut at the 200 &#956;m range may be adequate to visualize certain neuron types that can be contained within this slice thickness, for example pyramidal neurons in shallow layers of the cerebral cortex10,11,12, pyramidal neurons in the CA1 region of the hippocampus13,14, and granule cells in the dentate gyrus of the hippocampus15. Neurons with relatively longer dendritic&#160;trees, such as pyramidal neurons within deep layers of the cerebral cortex that for mouse can extend more than 800 &#956;m from the cell body16, provide a greater challenge because brains would need to be sectioned at a perfect angle to contain the entire dendrite tree within 200 &#956;m slices. This may not even be feasible if a dendrite or any of its branches extend in the rostral or caudal direction. While it is possible to address this limitation by tracing a neuron across multiple adjacent brain sections, this approach introduces a significant technical challenge in aligning the sections accurately for tracing17. A more practical approach would be to visualize entire neurons contained within brain sections that are cut at a greater thickness.
We report here a technique to stain neurons within 400 - 500 &#956;m thick brain sections of mice using the Golgi-Cox method, and to visualize their morphology using a high-resolution silicon oil-immersion objective that has an 800 &#956;m working distance. The Golgi-Cox impregnation and processing protocol that we describe is modified from one of the most-cited modern protocols in the literature6. Our approach with thick brain sections provides the advantage of increasing the probability of identifying neurons of any type that are fully contained within the section. In addition to the main protocol, we also present two variations that provide unique advantages: (1) Golgi-Cox staining with the cresyl violet counterstain on the surface of mounted sections, in order to define boundaries of brain regions and to identify layers of the cerebral cortex, and (2) Golgi-Cox staining with an intermediate freezing step for the long-term storage of impregnated whole brains prior to sectioning and final processing.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">potassium dichromate</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">P188-100</td>
			<td style="width:167px;">Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">potassium chromate</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">P220-100</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mercuric chloride</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">S25423</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Whatman grade 1 filter paper</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">1001-185</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">isoflurane</td>
			<td style="width:147px;">Pharmaceutical Partners of Canada</td>
			<td style="width:175px;">CP0406V2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">20 mL scintillation vial</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">03-337-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sucrose</td>
			<td style="width:147px;">Bioshop Canada</td>
			<td style="width:175px;">SUC700.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sodium phosphate monobasic</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">S5011-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">sodium phosphate dibasic</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">S9390-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">50 mL conical tube</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">12-565-271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">isopentane</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">AC126470010</td>
			<td>also known as 2-methylbutane. Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">agar</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">A1296-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">small weigh dish</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">02-202-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">vibratome</td>
			<td style="width:147px;">Leica</td>
			<td style="width:175px;">VT1000 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">6 well tissue culture plates</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">08-772-1b</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">mesh bottom tissue culture inserts</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">07-200-214</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:288px;">paraformadelhyde, 16%</td>
			<td style="width:147px;">Electron Microscope Sciences</td>
			<td style="width:175px;">15710-S</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">ammonium hydroxide</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">A669S-500</td>
			<td>Hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Kodak Fixative A</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">P7542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">superfrost plus slides</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">CitroSolv clearing agent</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">22-143-975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">anhydrous ethyl alcohol</td>
			<td style="width:147px;">Commercial Alcohols</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">cresyl violet</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:175px;">C1791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">permount</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:175px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">upright microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">BX53 model</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">colour camera, 12 bit</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">DV-47d</td>
			<td style="width:167px;">QImaging part 01-MBF-2000R-F-CLR-12</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:288px;">three-dimensional motorized microscope stage, controller and enoders</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">N/A</td>
			<td>Supplied and integrated with microscope by MBF Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">4x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">4x 0.16 N.A. UplanSApo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">10x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">10x 0.3 N.A. UPlan FL N&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">30x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td>30x 1.05 N.A. UPlanSApo&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">60x microscope objective</td>
			<td style="width:147px;">Olympus</td>
			<td>60x 1.42 N.A. PlanAPO N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">silicone immersion oil</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:175px;">Z-81114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Neurolucida software</td>
			<td style="width:147px;">MBF Biosciences</td>
			<td style="width:175px;">Version 10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">ImageJ software</td>
			<td style="width:147px;">U. S. National Institutes of Health</td>
			<td style="width:175px;">Current version</td>
			<td>With the OME Bio-Formats plugin installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Photoshop software</td>
			<td style="width:147px;">Adobe</td>
			<td style="width:175px;">version CS6</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neurons within thick brain sections using the golgi-cox method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

This Golgi-Cox staining protocol and its two described optional variants may be employed to visualize individual neurons within 400 - 500 &#956;m thick brain sections. Representative image montages of two-dimensional Z-projections captured using a 10X&#160;objective and 5 &#956;m steps in the Z axis are shown in Figure 1: A1 - C1&#160;for a large area of coronal brain sections that includes the anterior cingulate cortex area 1 and the secondary motor cortex<sup class="xre...

Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

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Neuroscience

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

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...