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>
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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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