Not long ago, Yacoin organized a full-blown experimental study of immunopathology, which aroused the interest of many curious babies. Everyone left messages, and the pathology experiment also included immunohistochemistry, pathology detection and other aspects, and there was still a lot of pathology knowledge that was not shown to everyone. Seeing the questions you have left, the editor will announce them one by one on the Yacoin WeChat platform in the follow-up. In this issue, the editor will start from the shallower to the deeper, starting with the knowledge of tissue slices for everyone.
When it comes to tissue sectioning, you will have many questions. Here is an analysis of your questions one by one:
- What is a tissue section?
A type of qie pian slide specimen. A thin slice of animal and plant tissues for optical or electron microscope observation. Due to different requirements, a blade can be used for freehand sectioning, or the tissue block can be embedded in paraffin or collodion or frozen at low temperature and sectioned with a microtome. Cut into 5~10 micron slices for observation by optical microscope. Ultra-thin sections cut with epoxy resin or methacrylic acid embedded tissue blocks, with a thickness of 20-50 nanometers, designed for observation under an electron microscope. The sections of root tips and stems used in general teaching are generally called paraffin sections.
- What is the initial shape of the tissue specimen of the tissue section?
The size of the tissue block: the ideal volume of the tissue block is 2.0cm×2.0cm×0.3cm, so that the fixative can penetrate into the tissue quickly and evenly. However, the tissue block is ideal according to the different preparation materials and purposes The volume is also different. For example, when making pathological examinations and scientific research sections, the tissue block can be thinned by 0.1~0.2cm, which can shorten the time of fixation, dehydration and transparency. If the thickness of the teaching section is 0.3~0.5cm, the same wax can be used. Block makes more teaching slices.
- How to make tissue dehydration transparent?
After the specimen is fixed and washed, the tissue contains a lot of water, and the water in the tissue block must be replaced. This process is called dehydration. Whether it is sliced with paraffin or collodion, the water in the tissue must be removed. Because water-containing tissue is incompatible with embedding materials such as paraffin wax and collodion, the commonly used dehydrating agent is a series of ethanol with different concentrations. .
The dehydration steps are: 80%, 90%, 95%, 100% ethanol of various concentrations for 2 hours, this process can be completed by the automatic control of the dehydrator.
Acetone is also a dehydrating agent with strong dehydration ability, but because of its strong dehydration ability, it has a violent contraction effect on tissues, so it is generally not used when making scientific research and teaching sections. Because ethanol, acetone, etc. are not soluble in paraffin, a solvent replacement process that can dissolve in paraffin is called transparent. Commonly used transparent agents are xylene, chloroform, methyl salicylate, etc.
- How to perform wax immersion, embedding, sectioning, and patching?
(1) Trimming wax block: Depending on the size of the tissue, cut off the remaining wax at about 0.1~0.2cm at the edge of the tissue, otherwise the tissue may shrink and become uneven.
(2) Prepare slicing tools: slicing knife, writing brush, ophthalmic tweezers (curved), bleaching temperature controller
(3) Install wax block: install the repaired wax block on a metal or wooden wax holder.
(4) Install the slicing knife: Install the slicing knife on the knife table of the slicing machine, and tighten the fastening screws on the knife table to prevent vibration during slicing and maintain a certain slice thickness.
(5) The thickness of the slice: the thickness regulator of the slicer is engraved with 0~50μm or 0~25μm, and the thickness can be selected arbitrarily. The thickness of the paraffin slice is generally 4~6μm.
(6) Slice
(7) Spreading: Use ophthalmic tweezers to lift the wax tape and gently spread it on the water surface at 40~45°C. Use the tension of the water and the temperature of the water to naturally flatten the slightly wrinkled wax tape.
(8) Patches and baking sheets: After the slices are fully flattened on the constant temperature water surface, the wax slices are pulled to the middle of the slide glass and the remaining water on the slide glass is poured, and placed in a 60-65 ℃ incubator Bake the slices in the oven of the temperature controller for 15 to 30 minutes to remove the paraffin that melts the tissue gap.
The picture shows a set of experimental equipment for paraffin sectioning: wax embedding machine, slicer, and spreader.
The picture shows the standard operation method of paraffin section。
- How to improve the level of tissue sectioning?
1) Pathology instrument is a necessary condition: choosing the right slicer is the primary factor of the experiment;
2) The quality of the sample to be cut is a key factor: the quality of the sample depends on the accuracy of the pre-processing work, including fixation, dehydration, embedding, etc.;
3) Model matching: suitable steel knife or disposable blade; carefully consider the sample type, size and thickness when slicing, taking into account the convenience and comfort of the operator;
4) The operator’s rich experience is essential: the operator must understand the working principle of the slicer and fine-tune the machine to achieve the best results; be able to deal with common failures and eliminate some potential influencing factors;
5) Elimination of common faults: such as the angle of the slicer blade, the temperature and hardness of the embedding block, whether there are notches or impurities attached to the blade, etc.
This issue mainly talks about the knowledge of tissue sectioning. Time is limited, so I have to say goodbye to everyone. At the same time in the next issue, there will be some tips on pathology experiments. Welcome everyone to leave a message and ask questions.
You are welcome to pay more attention to Abbkine’s official account, and there will be more scientific research surprises waiting for you. At the same time, in order to provide readers with a platform for learning biological knowledge, Abbkine’s technical experts have worked tirelessly to sort out the dry goods of a variety of high-end biological experiments, which will be published in the WeChat public account in the later period. Please continue to pay attention to Abbkine for readers who love science. No public.
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