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
<ol>
	<li>Golgi-Cox Impregnation of Brains
	<ol>
		<li>Make the Golgi-Cox solution of 1% (w/v) potassium dichromate, 0....

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The authors declare that they have no competing financial interests.

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 ...

The overall goal of this procedure is to determine the effective treatment on the morphology of neurons within the rodent brain. This method can help answer key questions in the neuroscience field, such as the normal and experimentally manipulated morphology of neurons within various regions of the rodent brain. This technique has the main advantage of increasing the probability of identifying and visualizing labeled neurons that are fully contained within single histological samples.Begin Golgi-Cox staining by placing a fresh mouse brain into a 20 milliliter glass scintillation vial containing 17 milliliters of Golgi-Cox solution. Protect from light and incubate at room temperature for 25 days. Once the incubation period has elapsed, Cryo protect the brain by placing it into 40 milliliters of sucrose cryoprotectant, and incubate in the dark at 4 degrees celsius for 24 hours.After the incubation in sucrose, the brain can be frozen for long term storage, or immediately blocked for sectioning as shown in the next section of the video. If freezing, immerse the whole brain in 200 milliliters of isopentane that has been precooled on dry ice. Then place the frozen brain into a 50 milliliter conical tube and store in the dark at minus 80 degrees celsius.To begin, remove the brain from the sucrose. And use a razor blade to block it for sectioning by cutting off the cerebellum, leaving a flat edge at the remaining caudal end of the brain. The brain must be blocked at a precise angle to ensure that neurons of interest are fully contained within the resulting sections.For example, we use a coronal plane that is perfectly perpendicular to the rostral caudal axis for cerebral cortex pyramidal neurons. Next, heat agar until it is melted. And let it cool until it is slightly above it's melting point.Then place the brain in a small disposable weigh dish with the caudal end face down. Add melted agar to cover the brain. Once the agar has solidified, trim the excess leaving approximately two to four milliliters surrounding the brain.Then use a small amount of ethyl cyanoacrylate to glue the brain to the stage of a vibratome with it's caudal end face down. Fill the stage area of the vibratome with enough sucrose cryoprotectant to cover the brain. Then section the brain at a slice thickness of 400 to 500 microns depending on the brain region to be examined.Using a small paintbrush, place brain sections into wells of a six well tissue culture plate containing mesh bottom inserts and pre filled with sucrose M phosphate buffer. Protect from light during sectioning. When finished sectioning, incubate sections in the dark at four degrees celsius overnight.Using the mesh bottom inserts, transfer the brain sections into a new well containing five milliliters of paraformaldehyde in phosphate buffer. Incubate on a rocker moving at slow speed at room temperature in the dark for 15 minutes. Wash the sections twice by transferring them to new wells containing five milliliters of water.Protect from light and wash at moderate speed at room temperature for five minutes. Next, transfer the sections into a new well containing five milliliters of ammonium hydroxide. Rock slowly in the dark at room temperature for 15 minutes.Wash the sections twice by transferring them to new wells containing five milliliters of water. Wash on a rocker moving at moderate speed in the dark at room temperature for five minutes. Transfer the sections into a new well containing five milliliters of diluted fixative A.Incubate on a rocker moving at slow speed in the dark at room temperature for 25 minutes.Wash sections in water twice as before. Use a small paintbrush to place the sections onto a microscope slide, then use tweezers and a small tissue to remove excess water and agar. Ensure that all agar is removed before proceeding with the dehydration steps.Allow the sections to air dry at room temperature protected from light. The appropriate drying time is critical, and should be determined for each laboratory. Too short of a time may result in sections falling off of slides during dehydration steps, and too long of a time may result in sections cracking.After drying, first place the slides in clearing agent for five minutes. Place the slides in 100%ethanol for five minutes. Then move through a series of decreasing ethanol concentrations for two minutes each.After the alcohol series, place the slides in water for five minutes. Then stain in 0.5%cresyl violet in water for seven minutes. Following the cresyl violet stain, place the slides in water for two minutes.Then dehydrate the sections through a series of increasing alcohol concentrations for two minutes each. Then two washes in 100%ethanol for five minutes. Place in clearing agent for five minutes.Finally add an anhydrous mounting medium and place a cover slip over the sections. Allow slides to dry horizontally in the dark at room temperature for at least five days. To capture high resolution image stacks of the area containing the neuron of interest, first select a 30X 1.05 NA silicone oil immersion objective on the microscope.Then apply three to four drops of silicone immersion oil to the slide. Place the objective over the slide, ensuring contact between the objective and oil. Focus the image and adjust the camera settings including the exposure and white balance in the imaging software.Next set the upper and lower boundaries for the image stack by focusing to the top of the section, and then selecting set next to top of stack within the image acquisition window. Then focus to the bottom of the section, and select set next to bottom of stack. Set the step distance to one micron by entering one micron next to distance between images within the image acquisition window.Lastly capture the image stack by selecting acquire image stack within the image acquisition window. Repeat the previous steps to capture the entire area of interest making sure that all image stacks overlap by at least 10%in the X and Y axes. This image of the cerebral cortex is a merged Z projection of image stacks acquired from a 500 micron thick section using a 10X objective.The tissue in this image was processed using the main all fresh protocol. The area indicated by the red square was imaged using a 30X silicon oil immersion objective and a higher resolution merged Z projection image stack was acquired. The red arrow shows the two dimensional Z projection of a neuron that was traced from the high resolution 30X image stack.Here the fresh protocol variant with cresyl violet staining was used. This higher resolution image shows the cresyl violet stained section. And this is the neuron tracing obtained.Finally this tissue was processed using the protocol variant in which brains are frozen following Golgi-Cox impregnation. Here the previously frozen tissue is seen at higher resolution. And once again a high quality two dimensional Z projection of the neuron tracing is obtained.This technique can be completed within one month with the processing and imaging steps completed within the final week. Brains can alternatively be frozen following Golgi-Cox impregnation allowing for the processing and imaging to be completed at a later date. Following this procedure, neuroscientists can use the captured images to perform detailed morphological analyses such as dendrite complexity or spine density in order to determine how an experimental manipulation can alter the structure of neurons within the brain.

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Research

JoVE Journal

Neuroscience

18.0K Görüntüleme.  University of Guelph. The overall goal of this procedure is to determine the effective treatment on the morphology of neurons within the rodent brain. This method can help answer key questions in the neuroscience field, such as the normal and experimentally manipulated morphology of neurons within various regions of the rodent brain. This technique has the main advantage of increasing the probability of identifying and visualizing labeled neurons that are fully contained within single histological samples.B...