Name: the cutting and floating method for paraffin-embedded tissue for sectioning
Uploaded: 2018-09-05T15:30:00
Description: Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

This work was supported by the National Natural Science Foundation of China (Grant No. 31400936, 31460260) and the Natural Science Foundation of Jiangxi Province of China (20171BAB215020). We also thank the joint program between Nanchang University and Queen Mary University of London for supporting this work.

All methods described here have been approved by the Animal Care and Use Committee of Nanchang University.
1. Assemble the Equipment and Connect the Microtome
<ol>
	<li>Design the parameter per the requirements (Supplementary Figure 1).</li>
	<li>Submit the parameter to a local factory to manufacture the acrylic boards.</li>
	<li>Assemble all parts in sequence: Use chloroform to combine 7 commercial acrylic boards into a tank with a section chan...

<ol>
	<li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li><li>Fujita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuron+differentiation+in+the+central+nervous+system+by+tritiated+thymidine+autoradiography.">Analysis of neuron differentiation in the central nervous system by tritiated thymidine autoradiography.</a> Journal of Comparative Neurology. 122 (3), 311-327 (1964).</li><li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+Computer-aided+jet-based+3D+tissue+engineering.">Organ printing: Computer-aided jet-based 3D tissue engineering.</a> Trends in Biotechnology. 21 (4), 157-161 (2003).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+tissues.">Cryosectioning tissues.</a> Cold Spring Harbor Protocols. 3 (8), (2008).</li><li>Viebahn, C., Luttenberg, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+anti-roll+plate+as+a+remedy+for+the+ill-effects+of+electrical+charge+during+cryosectioning.">A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning.</a> Journal of Histochemistry and Cytochemistry. 37 (7), 1157-1160 (1989).</li><li>Onozato, M. L., Hammond, S., Merren, M., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+completely+automated+tissue-sectioning+machine+for+paraffin+blocks.">Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.</a> Journal of Clinical Pathology. 66 (2), 151-154 (2013).</li><li>Sabaliauskas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+zebrafish+histology.">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li><li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid-Cut:+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples.">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754 (2016).</li><li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles.">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438 (2010).</li><li>Cornell, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+and+Thin+Sectioning+of+Microbial+Colony+Biofilms+for+Microscopic+Analysis.">Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.</a> Journal of Visualized Experiments. (133), e57196 (2018).</li><li>Whiteland, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+T-cell+subsets+and+other+leukocytes+in+paraffin-embedded+rat+and+mouse+tissues+with+monoclonal+antibodies.">Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 313-320 (1995).</li><li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification.">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (125), e55796 (2017).</li><li>Venegas-Pino, D. E., Banko, N., Khan, M. I., Shi, Y., Werstuck, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+and+Characterization+of+Atherosclerotic+Lesions+in+the+Murine+Aortic+Sinus.">Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus.</a> Journal of Visualized Experiments. (82), e50933 (2013).</li><li>Lau, S. K., Chu, P. G., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD163:+A+specific+marker+of+macrophages+in+paraffin-embedded+tissue+samples.">CD163: A specific marker of macrophages in paraffin-embedded tissue samples.</a> American Journal of Clinical Pathology. 122 (5), 794-801 (2004).</li><li>Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+Protocols+for+Human+Pancreatic+Islets.">Staining Protocols for Human Pancreatic Islets.</a> Journal of Visualized Experiments. (63), e4068 (2012).</li></ol>

To improve paraffin section morphology and solve the problem of wasted time during conventional paraffin sectioning, we created an improved paraffin sectioning method that combines cutting and unfolding. This improved method relies on simple equipment that comprises a section channel, a water bath and a heater with a temperature detection switch. The section ribbon enters the water bath through the section channel and unfolds automatically while cutting. Therefore, this method improves paraffin sectioning quality and eff...

Morphological study is important in the biological research. Although new technology has allowed researchers to observe their targets directly from the whole tissue or organisms1,2,3, cutting the specimen into thin sections, followed by staining, remains the primary method fornot only tissue morphology but also protein targeting directly in the tissue. Light microscopy uses three section types: paraffin, frozen, and semithin. Although cryosectioning is common for protecting tissue antigenicity, and the specimen preparation is simple, the retained tissue morphology is poor and unsuitable for thin sectioning4,5. Paraffin sectioning is the most frequently used method for exhibiting well preserved morphology. As the specimens are dehydrated completely and embedded in wax, the paraffin blocks can be stored indefinitely. In addition, paraffin sectioning produces thin sections that improve biological probe access in further experiments and reduce cell layer overlay in the Z direction.
However, conventional paraffin sectioning is tedious and demands operator skill. Paraffin sections undergo fixation, dehydration, embedding, cutting, and floating. Importantly, transferring section ribbons from the knife holder to the water bath is necessary but difficult for junior operators. Especially in dry air, the section ribbons will twist due to static electricity and are difficult to unfold on the warm water surface. To improve section quality, moistening the exposed tissue surface between microtome blade passes, cooling the wax blocks by immersing them in ice water, or raising the humidity with a humidifier near the microtome are recommended6,7. Newer methods for improving paraffin sectioning include hybrid paraffin embedding, cryosectioning8, and commercial section transfer system assistance9. Although these methods partially improve paraffin sectioning speed and quality, they make sectioning much more cumbersome, and commercial section transfer systems are expensive.
In this protocol, we demonstrate how to create simple, cheap and flexible equipment step by step, which can be connected to the blade holder of a rotary microtome. This equipment is comprised of a section channel, a water bath, and a heater with a temperature detection switch. After cutting, dozens of sections flow into the section channel and enter the water bath directly, thus unfolding automatically. This improves the efficiency of paraffin sectioning and makes this technology more convenient. Using this method, more adult mouse hippocampal sections, adult mouse kidney sections, embryonic 15.5 day-old (E15.5) mouse brain sections, and adult zebrafish eye sections were harvested in less time and remained more intact morphologically. This method can also be used for other tissue samples that require accelerated paraffin sectioning while avoiding loss of section distinction.

<table>
	<tbody>
		<tr>
			<td>Incubator</td>
			<td>Boekel Scientific</td>
			<td>133000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Module</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial acrylic board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubular electric heating element(12V 200W)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller(12v 120w)</td>
			<td>Mingsuo</td>
			<td>XH-W3002</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary microtome&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral silicone sealant</td>
			<td></td>
			<td></td>
			<td>Link the water channel with the microtome knife holder</td>
		</tr>
		<tr>
			<td>Voltage transformer</td>
			<td>Dearll</td>
			<td>S-250-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable blade</td>
			<td>Accu-Edge</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Baso Diagnostics Inc.</td>
			<td>BA-4025</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslide</td>
			<td>Sail Brand</td>
			<td>7105</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sinopharm Chemical Reagent Co.,Lid</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip&#160;</td>
			<td>Citoglas</td>
			<td>10212424C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Xilong Chemical</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath for paraffin sections</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCore Arcadia C&#160;- Cold Plate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin repellent spray&#160;</td>
			<td>Thermo Scientific</td>
			<td>9990420</td>
			<td></td>
		</tr>
	</tbody>
</table>

the cutting and floating method for paraffin-embedded tissue for sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

The improved method increased the number of intact paraffin sections. We tested this new method on adult mouse hippocampal tissue, adult mouse kidneys, embryonic mouse brains, and zebrafish eyes. Water was added to the tank, and the water temperature was maintained between 38.0 &#176;C to 40.0 &#176;C. After a serially preparing the tissue samples, they were sectioned and compared to conventional sectioning. The new method avoided section loss and increased the proportion of intact sectio...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

This method can help answer key questions in morphological field, such as paraffin sectioning. The main advantage of this technique is that culminates cutting and the folding procedure and avoid transfer process that required by conventional method. To begin, gather all of the pre-manufactured parts in a fume hood.Use chloroform to combine the seven acrylic boards, as shown here. The end product will be a tank equipped with a section channel and a water bath. Install the tubular electrical heating element through the hole in acrylic board number two.Using an electric drill, make a small hole in acrylic board number seven, and install a digital thermostat on the tank, making sure that it is at least one centimeter below the level of the intended water surface, which is indicated by the black dot on the tank. Adjust the voltage so that it is below 24 volts before turning on the equipment. Remove the section waste tray from the microtome.Next, use neutral silicone sealant to link the sections channel to the microtome blade holder, making sure not to get any sealant above the top of the blade. Let the setup dry overnight to give the chloroform and neutral silicone sealant time to solidify. The next day, install the blade in the blade holder.Add water to the water bath, making sure that the water surface just touches the top of the blade. After performing conventional paraffin sectioning, turn on the equipment to begin improved paraffin sectioning. Use the temperature detection switch to open the heater.Set the targeted temperature between 38 degrees Celsius and 40 degrees Celsius for one hour to warm the water. Use a knife to modify the paraffin block acquired by paraffin embedding. Then, place the block on the microtome's cassette clamp, and lock it.Cut the thicker sections to quickly approach the sample. Adjust to a suitable section thickness for the samples. Turning the hand wheel will automatically load the sections into sections channel and bath.Next, section the specimen in a slow and uniform cutting stroke. Guide the sections into the bath chamber so that they float in a line. If the head section adheres to the chamber wall, use a brush guide to keep it floating.After the sections fully unfold in the water bath, use tweezers to separate several, and then pick them up on a microscope slide. Transfer the slides to an oven set to 42 degrees Celsius to dry overnight. The slides can then be stored at room temperature indefinitely until ready to analyze.When ready to analyze, use hematoxylin-eosin staining to evaluate the sections using the improved method. In this study, an improved method is used to prepare paraffin sections for adult mouse hippocampal tissue, adult mouse kidneys, embryonic mouse brains, and zebrafish eyes. As shown here, the new method avoids section loss and increases the proportion of intact sections in each type of examined tissue.When performing whole-series sectioning, this method is seen to accelerate the paraffin sectioning speed in all tested samples. In other words, this method provides higher quality paraffin sectioning in less time than the conventional method. Series sections of zebrafish optic retina are then observed by hematoxylin-eosin staining.The sections harvested from the improved method are seen to have better integrity than those harvested from the conventional paraffin sectioning method. While there are no significant differences in the quality of most harvested sections when comparing these two methods, some sections harvested from the conventional method are seen to have relatively low quality. Don't forget that working with chloroform can be extremely hazardous, and precautions such as fume hood should always be taken while performing this procedure.While attempting this procedure, it's important to remember, add water carefully and avoid overfill.

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Research

JoVE Journal

Neuroscience

Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of this cellular mosaic often requires that the organ of Corti is cross-sectioned. In particular, the nonsensory pillar and Deiters' cells, whose nuclei are located basally with respect to the hair cells, cannot be visualized without cross-sectioning the organ of Corti. However, the delicate cytoarchitecture of the mammalian organ of Corti, including the fine cytoplasmic processes of the pillar and Deiters' cells, is difficult to preserve by routine histological procedures such as paraffin and cryo-sectioning, which are compatible with standard immunohistochemical staining techniques.
Here I describe a simple and robust procedure consisting of vibratome sectioning of the cochlea, immunohistochemical staining of these vibratome sections in whole mount, followed by confocal microscopy. This procedure has been used widely for immunhistochemical analysis of multiple organs, including the mouse limb bud, zebrafish gut, liver, pancreas, and heart (see 3-6 for selected examples). In addition, this procedure was sucessful for both imaging and quantitificaton of pillar cell number in mutant and control organs of Corti in both embryos and adult mice 7. This method, however, is currently not widely used to examine the mammalian organ of Corti. The potential for this procedure to both provide enhanced preservation of the fine cytoarchitecture of the adult organ of Corti and allow for quantification of various cell types is described.

A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types.

The mammalian organ of Corti is a highly ordered cellular mosaic of mechanosensory hair and nonsensory supporting cells (reviewed in 1,2).Visualization of ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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 validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

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

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

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

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the diagnosis still relies only on clinical signs of motor involvement that appear later on in the disease course, when most of the dopaminergic neurons are already lost. Hence, there is a strong need for a biomarker that can identify patients at the beginning of disease or at the risk of developing it. Over the last few years, skin biopsy has proved to be an excellent research and diagnostic tool for peripheral nerve diseases such as small fiber neuropathy. Interestingly, a small fiber neuropathy and alpha synuclein (&#945;Syn) neural deposits have been shown by skin biopsy in PD patients. Indeed, skin biopsy has the great advantage of being an easily accessible, minimally invasive and painless procedure that allows the analysis of peripheral nervous tissue prone to the pathology. Moreover, the possibility of repeating the skin biopsy in the course of the follow-up of the same patient allows studying the longitudinal correlation with the disease progression. We set up a standardized reliable protocol to investigate the presence of &#945;Syn aggregates in skin nerve fibers of the PD patient. This protocol involves few short fixation steps, a cryotome sectioning and then a free-floating immunofluorescence double-staining with two specific antibodies: anti Protein Gene Product 9.5 (PGP9.5) to mark the cutaneous nerve fibers and anti 5G4 for detecting &#945;Syn aggregates. It is a versatile, sensitive and easy to perform protocol that can also be applied for targeting other proteins of interest in skin nerves. The ability to mark &#945;Syn aggregates is another step forward to the use of skin biopsy as a tool for establishing a pre-mortem histopathological diagnosis of PD.

Here, we present a protocol for a free-floating indirect immunofluorescence assay on skin biopsy sections that allows for the identification of disease specific conformation variants of alpha synuclein involved in Parkinson disease and multiple proteins of the peripheral nervous system.

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...