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.
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.
