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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;

1. Perfusion and tissue processing
<ol>
	<li>Anesthetize mice using an intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). Pinch toes to confirm a lack of response before continuing.</li>
	<li>Transcardially perfuse with ~50 mL of ice-cold 0.1M Dulbecco&#39;s phosphate-buffered saline (DPBS) followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB). 
	&#160;CAUTION: PFA is toxic. Use personal protective equipment (PPE) when working with PFA.</li>
	<li>Remove the brains and immerse them in 4% PFA in 0.1 M PB at 4 &#176;C for 24 h for post-fixation. Wash with DPBS 3x.</li>
	<li>Change to 15% sucrose solution in 0.1 DPBS overnight and then 30% sucrose solution in 0.1 M DPBS for another 48 h at 4 &#176;C.</li>
	<li>Remove the tissue from the sucrose solution and freeze in a cryotome chamber preset to a temperature of -20 &#176;C. Frozen samples can be stored in sealed tubes at -80 &#176;C until sectioning.</li>
	<li>Embed tissues in optimum cutting temperature embedding medium (see Table of Materials) and mounted on a specimen disc. Cut serial 30 &#181;m thick sections in a coronal plane using a cryotome and collect all the sections consequently in 24 well culture plates filled with cryoprotectant solution (30% glycerol, 30% ethylene glycol, 40% DPBS; Figure 1A&#8211;C). Store in a -20 &#176;C freezer. 
	&#160;NOTE: Thicker sections (e.g., 50 &#181;m) are preferable when feasible. Make sure that the sections are kept in cutting order and that all sections are completely in cryoprotectant. A 96-well plate can be used as an alternative to 24 well plates to collect sections.</li>
	<li>Cover the wells with plate sealer to prevent evaporation and drying. Store plates at -20 &#176;C until further use. Sections stored at -20 &#176;C are stable for several months for choline acetyltransferase (ChAT) immunohistochemistry.</li>
</ol>
2. Systematic section selection for IHC
NOTE: A pilot study should be done to know the total number of sections required to achieve an acceptable coefficient of error (CE). The CE value is an expression of the total amount of error in the sampling procedure. The lowest value represents the minimal error, and a CE value lower than 0.1 is considered acceptable by the software used here (see Table of Materials).
<ol>
	<li>Identify the first and last sections for each brain by comparing morphological features with a standard mouse brain atlas such as Franklin and Paxinos. Nucleus basalis of Meynert or NBM begins at bregma -0.0mm and ends at -1.6 mm. Therefore, about 50 sections contain NBM. The total number of sections is one of the parameters required during stereology. The selection of every 8th section (SSF = 1/8) gave a total of 6&#8211;7 sections for the analysis and yielded an acceptable CE for both volume estimation and fiber length estimation in our pilot study.</li>
	<li>Randomly start with one of the first eight sections and then systemically sample every 8th section until the last posterior section containing NBM (Figure 1C).</li>
</ol>
3. Immunohistochemistry
<ol>
	<li>Transfer the cryopreserved sections to room temperature in cryoprotectant and then 0.1M phosphate buffer (PB) in 6 well plates.</li>
	<li>Wash 3x in PB.</li>
	<li>Incubate in 0.3% H2O2 in methanol for 15 min.</li>
	<li>Wash 3x in Tris-buffered saline (TBS).</li>
	<li>Incubate in 0.25% Triton X-100 in TBS for 30 min.</li>
	<li>Block with 10% normal bovine serum (NBS) in 0.1% Triton X-100 in TBS for 30 min.</li>
	<li>Incubate with a 1:1,000 dilution of goat anti-human ChAT primary antibodies (see Table of Materials) in TBS with 0.1% Triton X-100 and 1% NBS for 48 h at 4 &#176;C.</li>
	<li>Wash 3x in TBS at room temperature. Perform all further incubation and washing at room temperature.</li>
	<li>Incubate with biotinylated bovine anti-goat secondary antibody (see Table of Materials) for 1 h.</li>
	<li>Wash 3x in TBS.</li>
	<li>Incubate with the avidin-biotin&#8211;peroxidase complex (see Table of Materials).</li>
	<li>Wash 3x in TBS.</li>
	<li>Develop using an enhanced DAB peroxidase substrate solution (see Table of Materials) according to the manufacturer&#39;s recommendations. 
	&#160;CAUTION: DAB is a suspected carcinogen. It is toxic by contact and inhalation. Use PPE when working with DAB.</li>
	<li>Wash the section a couple of times with distilled water and keep in Tris pH = 7.6.</li>
	<li>Mount the sections on the gelatinized slides. All sections from one tissue can be put on the same slide. Air dry the sections, dehydrate 2x for 5 min each with 70%, 90%, 95%, and 100% ethanol, and then clear the sections with two 10 min xylene washes. Coverslip the sections using a mounting medium (see Table of Materials). 
	&#160;NOTE: The processing time of dehydration and clearing affects the thickness of the sections. Therefore, the same conditions should be used for all sections. In the current study, the mean value for the final thickness was 21.11 &#177; 0.45 &#181;m.</li>
	<li>Keep the sections in the fume hood to dry. The dry sections are ready for stereology.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABC kit</td>
			<td>Vector Laboratories</td>
			<td>PK6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ChAT Antibody</td>
			<td>Millipore, MA, USA</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine anti-goat IgG-B</td>
			<td>Santacruz Biotechnology</td>
			<td>SC-2347</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum, Adult</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Lieca Microsystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>D5652</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>324558</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Immpact-DAB kit</td>
			<td>Vector Laboratories</td>
			<td>SK4105</td>
			<td>Enhanced DAB peroxidase substrate solution</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Westward Pharmaceuticals, NJ, USA</td>
			<td>0143-9509-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Lieca Microsystems, Buffalo Grove, IL, USA</td>
			<td>AF6000</td>
			<td>Equipped with motorized stage and IMI-tech color digital camera</td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (O.C.T.) embedding medium</td>
			<td>Electron Microscopy Sciences, PA, USA</td>
			<td>62550-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Electron Microscopy Sciences, PA, USA</td>
			<td>17986-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T1503</td>
			<td>Tris base</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>T5941</td>
			<td>Tris hydrochloride</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>Rompun</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Histological grade</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemical staining of nerve fibers in the nucleus basalis of meyner of the mouse brain

Source: Singh, P., et al. <a href="https://app.jove.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

<img alt="Figure 1" src="/files/ftp_upload/23194/23194_Figure1.jpg" /> 
Figure 1: Illustration of tissue processing and sampling used in the present study. Sample preparation and sampling. (A) The cerebellum and olfactory bulb are removed before mounting on the specimen disc. (B) Orientation of the brain on the specimen disc for section cutting. A shallow longitudinal incision (marked with a line) on the one hemisphere helps to identify the side of the brain in the s...

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...

Take a fixed mouse brain section containing the nucleus basalis of Meynert, rich in nerve fibers expressing the enzyme choline acetyltransferase.
The section is pretreated to deactivate endogenous peroxidase.
Add a detergent to permeabilize cellular membranes, followed by a blocking solution to prevent non-specific antibody binding.
Replace the solution with primary antibodies that bind to choline acetyltransferase.
Wash with buffer to remove unbound antibodies. Incubate with biotin-tagged secondary antibodies, which bind to the primary antibodies.
Remove unbound antibodies and add avidin-biotin-peroxidase complexes, which bind to the secondary antibodies.
Wash to remove excess complexes.
Add a chromogenic substrate that reacts with peroxidase, forming a colored precipitate.
Rinse with distilled water to remove excess precipitate.
Transfer the section onto a gelatinized slide.
Next, dehydrate the section using increasing ethanol concentrations, followed by xylene.
Mount the section and observe under a microscope to visualize the stained nerve fibers.

immunohistochemical-staining-nerve-fibers-nucleus-basalis-meyner

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19 조회수. Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020) This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary...

비디오: Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain - 개념

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

재조합 모터 영역의 광섬유 연결 : 광섬유 해부, DTI 및 3 차원 문서화 방법론

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

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JoVE Journal

신경과학

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

광학 Fractionator 표준 현미경 장비를 사용 하 여 마우스 Substantia Nigra에서 Dopaminergic 신경 수 stereological 추정

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Section(신선 냉동 또는 고정 마우스 뇌 절편에서 Multiplex Fluorescence In Situ Hybridization과 형광 면역조직화학의 결합)

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

내레이션 대본

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

재조합 모터 영역의 광섬유 연결 : 광섬유 해부, DTI 및 3 차원 문서화 방법론

광학 Fractionator 표준 현미경 장비를 사용 하 여 마우스 Substantia Nigra에서 Dopaminergic 신경 수 stereological 추정

Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Section(신선 냉동 또는 고정 마우스 뇌 절편에서 Multiplex Fluorescence In Situ Hybridization과 형광 면역조직화학의 결합)