The detailed process and precautions of paraffin sectioning

The steps to make paraffin sections are as follows:

1. Tissue embedding.

1) Ethanol dehydration: the tissue is dehydrated step by step by different concentrations of ethanol solution, and all levels of ethanol are used for 40min (note: bony and calcified tissues need to be soaked in JYBL- decalcification solution for 24 hours, and then ethanol dehydration steps are carried out after the tissue is softened);

2) Transparency: The tissue is soaked in three dimethyl benzene sequentially, each cylinder for 1h;

3) Wax immersion: The tissue is soaked in three paraffin cylinders in turn, each for 1 h;

4) Embedding: pour the liquid paraffin wax into the mold box, then lay the tissue block soaked in wax at the bottom, pay attention to the direction of the cut surface and place it downward, remove the embedding frame after the paraffin solidifies, and then trim the wax block after it is completely cooled and hardened, and the paraffin around the tissue is retained moderately for sectioning.

2. Slice production: fix the pre-cooled wax block on the paraffin microtome, make the cutting surface of the wax block parallel to the knife edge, and the inclination of the knife is usually 15, rotate the rotary thruster, adjust the thickness of the slice to 4 m, and cut it into slices of uniform thickness.

Hold the brush in your left hand, rotate the slice with your right hand and take the machine to turn it, after the slice is brought out, gently hold it up with the brush, and then use tweezers to lightly tweezer the wax piece, and put it into the exhibition box with the front, and its water temperature is about 45. After flattening, remove the slices. Attach the slice with the left hand to hold one end of the slide, enter the water vertically to attach the slice, and use tweezers to push with the right hand to attach two-thirds of the slide.

After the slices are attached, they are placed in the air to dry slightly, placed on a 65 baking machine for 1 hour, and then placed in the oven for 2 hours.

3. Deparaffinize paraffin sections to water.

In turn, the paraffin sections were put into xylene, 10min-xylene, 10min-xylene, 10min-anhydrous ethanol, 5min-absolute ethanol, 5min-90% alcohol, 5min-80% alcohol, 5min-70% alcohol, 5min-50%, 5min-50%, alcohol, 5min gradient dewaxing.

4. HE staining.

Paraffin sections were stained with hematoxylin for min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, then returned to blue with 1% ammonia aqueous solution for 1min, rinsed with running water for a few seconds, dyed in eosin staining solution for a few seconds, and rinsed with running water.

5. Dehydration seal.

The paraffin sections were sequentially put into 75% ethanol 2min -85% ethanol 2min - absolute ethanol 5min - absolute ethanol 5min - xylene 5min transparent, and the slices were taken out from xylene and covered with neutral gum.

6. Interpretation of results.

The nucleus is blue and the cytoplasm is red.

The paraffin sectioning method includes steps such as material extraction, fixation, washing and dehydration, transparency, wax dipping, embedding, sectioning and patching, deparaffinization, staining, dehydration, transparency, and mounting.

Material: The material must be fresh, and if it is left for too long, it will cause protein decomposition and denaturation, resulting in cell autolysis and bacterial growth, but cannot reflect the morphological structure of the tissue when it is alive.

Fixation: The fixative solution is impregnated with fresh material cut into small pieces, maintaining as much as possible its structure when it was alive. The amount of fixative solution is usually about 20 times that of the material block, and the fixation time depends on the size and looseness of the material block and the penetration speed of the fixative solution, which can range from 1 hour to several days, usually 1 hour to 24 hours.

Washing and dehydration: After fixation, the fixative solution and its crystallization precipitate left in the tissue should be removed, otherwise the staining effect will be affected in the later stage. Alcohol is a commonly used dehydrating agent, and in order to reduce the sharp shrinkage of tissue material, it should be carried out in an increasing sequence from low to high concentration, usually starting with 30% or 50% alcohol and working through % until pure alcohol (absolute ethanol) for 1 hour at a time.

Transparency: Commonly used clarifying agents are xylene, benzene, chloroform, n-butanol, etc., and all kinds of clarifying agents are solvents for paraffin. The impregnation time of the clarifier depends on the size of the tissue material and whether it belongs to the cyst cavity or solid organ.

If the transparency time is too short, the transparency is not complete, and it is difficult for paraffin to immerse into the tissue; If the transparency time is too long, the tissue will harden and become brittle, and it will be difficult to cut out the whole section, which can last up to several hours.

Wax impregnation and embedding: Usually the tissue material block is immersed in the same amount of molten paraffin wax and xylene mixture for 1 2 hours, and then moved into 2 molten paraffin liquid for about 3 hours, and the wax impregnation should be carried out in a temperature chamber higher than the paraffin melting point of about 3 to facilitate paraffin immersion into the tissue. The wax-impregnated tissue material blocks are placed in a container filled with wax solution to make wax blocks.

Sectioning: The prepared wax block is mounted on a paraffin microtome such as a Revolde S700 sample head for sectioning, usually with a slice thickness of 3 8um. The cut slices are rolled up with a brush and gently spread out in a spreader with a water temperature of about 45.

Patches and baking sheets: Break a certain length of wax strip (continuous sectioning) or use tweezers into a single wax piece, flatten it, and then remove it to the slide and put it in a 45 incubator to dry, or dry in a 37 oven, but it needs to be extended appropriately.

Section deparaffinization and hydration: Dried sections need to be deparaffinized and hydrated to stain in a water-soluble stain. Dewax with xylene.

Staining: The goal is to make different structures within the cell tissue appear different colors for easy observation.

Slice dehydration, transparency and mounting: The stained section needs to be dehydrated by gradient alcohol, and the time in 95% and pure alcohol can be appropriately extended to ensure complete dehydration; If the dye is prepared with alcohol, the time in the alcohol should be shortened to avoid decolorization. After xylene is transparent, quickly wipe off the excess liquid around the material, add an appropriate amount (1 2 drops) of neutral gum dropwise, and cover the slide with a coverslip.

You can ask Reward customer service for specific tutorials or something, and they usually give them.

Paraffin sections. The production process mainly includes: material extraction, fixation, dehydration, transparency, wax penetration, embedding, slicing, patching, dyeing, transparency, sealing and other steps.

1 Ingredients. The quality of the material directly affects the quality of the slice, no matter which animal and plant material is taken, the following points must be noted.

1) The selection of plant materials must not damage the plant body or the required part as much as possible; Animals are often anesthetized when animal materials are used, and anesthetics are commonly used.

There is chloroform and ether, or the desired tissue is quickly removed after the animal is killed.

2) The material must be fresh, which is essential for those engaged in cell biology.

It is particularly important to study that living tissue blocks should be removed as much as possible and then injected with fixative solution.

3) The knife should be sharp when cutting the material to avoid damage due to squeezing the cells.

4) The cut material should be small and thin, so that the fixative can quickly penetrate into the inside. Generally, the thickness does not exceed 2mm, and the size does not exceed 5 5mm2.

2 Fixation. After tissues and cells leave the body, they continue to live for a certain period of time, causing pathological changes until they die. In order for the specimen to reflect its normal state during its lifetime, it is necessary to quickly kill tissues and cells with certain chemicals as soon as possible, inhibit these changes, and convert structural components into insoluble substances to prevent the dissolution and disappearance of certain structures.

This treatment is fixed. In addition to the above effects, fixatives harden the tissue properly for subsequent processing, alter the refractive index inside the cell and make certain parts easy to stain.

The fixative is mainly used for proteins, and other components such as fats and sugars are not considered in general production, and if these substances are to be observed, they can be fixed in a special way.

The effect of the fixative is manifested in the change of the volume of the material, the degree of hardening, the speed of penetration, and the effect on staining. The quality and magnitude of these effects depend on the nature of the material being fixed, and the same fixative solution is good for one material, but may not be very suitable for other tissues. The characteristics that a good fixative must have are:

The speed of penetration into tissues is fast. It can coagulate the inclusions in the cell into insoluble substances, do not make the tissue swell or contract to maintain its original shape, the degree of hardening of the tissue is moderate, increase the refractive power of the cell inclusions, increase the ability of mordant and staining, and have the effect of a preservative. Fixatives are divided into simple fixatives and mixed fixatives.

A simple fixative is a single fixative, and ethanol is commonly used.

Formaldehyde, glacial acetic acid.

Mercury, picric acid, chromic acid, potassium dichromate and osmium acid. Among them, picric acid, mercury and chromic acid can coagulate cytoalbumin.

It can also coagulate nucleoproteins; Ethanol can only coagulate albumin, while acetic acid can only coagulate nucleoprotein; Formaldehyde, hungry acid, and potassium dichromate do not coagulate for either protein.

In teaching, under the light microscope.

Most of the observation section specimens were prepared by paraffin sectioning. After leaving the body, the tissue will soon die and cause tissue decay, and lose its original normal structure, so the tissue should be fixed, paraffin-embedded, sectioned and stained to avoid the death of the cell tissue, and its morphological structure can be clearly identified.

Advantages: 1. The morphology of tissue cells is clear.

2. The sections can be stored for a long time for teaching, scientific research, pathological diagnosis and review, and can be used for retrospective research of other projects.

3. It can be cut (2 3 microns) of thin slices, ** thin and uniform, without wrinkles.

Disadvantages: 1. There are many paraffin wax production procedures and links, and it takes several days to complete one cycle.

2. It is easy to cause the loss of antigenicity in the tissue during the production process, which affects the sensitivity of the results when used for immunohistochemical staining.

Paraffin sections.

Baiparaffin Section) is the most widely used method of DAO in conventional DU production techniques. Paraffin sections are not only used to observe the morphological structure of normal cell tissues, but also the main method used to study, observe and judge the morphological changes of cell tissues in pathology and forensic science, and have also been widely used in many other disciplines.

The paraffin sectioning method includes steps such as material extraction, fixation, washing and dehydration, transparency, wax immersion, embedding, sectioning and adhesive slice, deparaffinization, staining, dehydration, transparency, and mounting. Generally, it takes several days from the fixation of the tissue to the sealing of the slide, but the specimen can be stored for a long time and is a permanent microslide specimen.

1.The microtome should be placed smoothly, and the slicing knife and wax block should be installed firmly, otherwise the slice will be wrinkled or uneven in thickness due to vibration.

2.When slicing, the knife edge should be cleaned in time and the wax chips should be removed, otherwise it is easy to cause the slice to break.

3.The inclination angle between the slicing knife and the cutting surface of the wax block should be 5 degrees to 10 degrees, and if it is too large, the slice will be rolled up, and it is not easy to connect the cracked rubber into a wax belt; If it is too small, it will be wrinkled.

4.When slicing, the speed of shaking the rotating wheel should not be too fast, and the force should be uniform and stable.

5.The water temperature during the exhibition is between 42 degrees and 48 degrees, and 45 degrees is optimal.

6.Clean wax chips and other debris in the water in time to prevent contamination of the sections.