Precautions General sliced

Precautions General sliced 

1. Drawn 
     Drawn will directly affect the quality of the slice. Doctors in the drawn first to have drawn a sharp knife, cutting tissue 
To avoid dragging drawn knife back and forth, tissue blocks were cut to a uniform thickness, generally 0.2 ~ 0.3cm thickness as appropriate, brittle hair easier to group 
Organizations such as the thyroid, liver, blood clots, lymph nodes, and other tissues may be appropriate chunks a little thick, and adipose tissue, lung tissue, fibrous tumor, 
Leiomyoma or reagents difficult to penetrate dense tissue should be taken thinner; lymph nodes on both sides of the ball off the crown should be revised, and try to eliminate fat around 
Fat tissue. In the drawn great attention to whether there should also sutures or bone tissue within the organization, such as the inevitable encounter calcified tissues, and technology should be 
Room made it clear, in the cut fibrous tissue, muscle tissue, gastrointestinal tract, and muscle fiber should pay attention to, when drawn as much as possible by fiber 
Parallel to cut better. 
     2 Fixed 
     Fixed is the first technical room work, but also in the whole process can not be remedied producers step. The so-called "fixed", is to organize 
After in vitro, using a variety of ways to make the material as close to the process within the cell morphology and location of their living state. The purpose of the fixed 
Is to prevent tissue autolysis and corruption, preventing the decomposition of the enzyme protein within the cell, so that the various components of cells such as eggs 
White, fat, carbohydrate or enzymes into the insoluble material to maintain the original structure and life similar. In addition, solid organization 
After the set, showed some hardened state, increased tissue toughness, and easy to distort, after tissue processing favor. So once the organization 
Necessary for timely vitro fixed amount fixative should be five times the organization. Fixed principal and fixed key timeliness, fixative choice 
Concentration of fixative, fixed temperature and time dependent. The most common fixative is 10% formalin. I believe that in 10% formalin 
Due to the quality of formalin, and volatilization when drawn into the water on the concentration used is insufficient lowering of tissue fixation, and 
High concentrations of formalin, reducing the tissue penetration, formed around the central fixation than fixed phenomenon. With practice, this 
Considered to 12 to 15% acid formalin best. Large specimens (2cm thick) generally requires 6 to 12 hours, generally require small samples 
3 to 6 hours; When low temperature 18 ℃, 37 ℃ is available at the following temperature inside the heat 4 to 6 hours. Because HE staining best coloring PH 
3.5 to 4.5, neutral formalin have some impact on hematoxylin staining nuclei easily gray, poor nuclear chromatin. 
     So, although neutral formalin action against the original well preserved, but the vast majority of antigens acid Formalin is also very 
Good preservation effect, so no special handling in the case of the conventional HE acid with 12 to 15% formalin may be (per 100 ml 
Add 12 to 15% formalin solution 5 ml of glacial acetic acid). Bone, connective tissue fixed in Bouin solution is better, were fixed by Bouin after 
Organizations must running water for more than 4 hours. Conditional units can be used two or more than two kinds of fixative according to different requirements; minority single 
Bit can also create an organization freezer to accommodate a variety of special handling requirements. 
     3. Washing 
     After fixing water (10 to 20 minutes), is often overlooked by many people, but not completely washing, staining likely to cause off-chip and not bright. 
Immunohistochemical to be done on the organization, but should not be overlooked. Formalin is not conducive to the preservation of tissue antigens. 
     4 dehydration and transparent 
     Dehydration is the use of so-called dehydrating agent to displace the water within the organization, in order to facilitate penetration of the organic solvent, a process called dehydration. 
Dehydration is complete, is directly related to whether the organization can fully transparent, and excessive dehydration (due to tissue in pure alcohol for too long, hair 
Health Organization texture changes) is likely to cause tissue brittle. Brittle tissue causes heating there (the temperature exceeds 35 ℃), a dehydrating agent 
With added acetone, xylene, paraffin wax dip excessive use and so on. We always thought that the organization generally crisp with a high concentration of alcohol-related, while ignoring 
The relationship between low levels of alcohol, in fact, a low concentration of alcohol has a strong penetration, more easily than pure alcohol to penetrate into tissue inside so 
Dehydrated, if the tissue sufficiently dehydrated alcohol in a low concentration, high concentration of alcohol in the removal of water from only a low concentration to a high concentration of between 
Therefore, only a short time to complete, if the time does not shorten the time of pure alcohol, it will cause tissue crisp; If dehydration heating, 
Acetone, can also cause tissue contraction and affect immunohistochemical markers. For dipping into the paraffin tissue blocks must 
A mixture of both with a dehydrating agent, but also a medium compatible with the wax material, this process is called transparent. Transparent agent is currently the best-xylene. 
Many people think that xylene very easy to organize crisp, this view is very one-sided, at room temperature, if it is not too long (more than 24 hours 
Tissue above) does not cause tissue xylene too brittle hair, on the contrary make some organizational structure is relatively tough quality: for example, such as uterine fibroids 
Pretty good cut), but when the temperature exceeds 35 ℃ after xylene, can easily cause tissue brittle. So I think the best of the size of the specimen 
Open dehydration, under normal circumstances, a large specimen dehydration time (room temperature 18 ℃) 2 hours with 80% ethanol, 95% ethanol (2) one half the 
Hours to 2 hours of pure alcohol (3) each a half hour to two hours, and xylene (2) each 30-60 minutes is appropriate; small specimens off 
Water time should be reduced accordingly. Dehydration duration is closely related to the temperature level. We found that in practice, room temperature 12 ~ 15 ℃ 
When, as a critical temperature range. When the temperature is higher than this temperature range, easily dehydrated tissue, the dewatering time can be shortened; whereas 
When room temperature is lower than the temperature range should be extended drying time (such as to ensure a constant temperature at best). Replacement of the reagent should be 
Volume-based, quantitative replacement, can not wait to go to replace a bad slice. Otherwise, there will be at least 2 to 3 batches biopsy bad. According to my 
Branch experience, such as the amount of reagent 500ml, per 500 block of wax once a suitable replacement. 
     5. Baptist wax 
     After the tissue transparent, dipping in melted paraffin wax process called leaching. Soaked with other agents (fiery cotton, gelatin, etc.) into groups 
The process may be referred to the internal organization or impregnated penetration. Wax dipping temperature is too high (above 60 ℃), xylene was added in the wax or wax due zona 
Excessive intake of xylene, will lead the organization stiff, but also the destruction of the antigen within the organization; the authors advocate using paraffin wax melting dip 
Point at 56 ℃ (three), first: Paraffin 30 minutes; Second: Paraffin 90 minutes; Third Road: paraffin, 90 minutes. Dip warm wax 
Controlled at around 56 ~ 58 ℃. (First 1:3 can also be used paraffin wax dipped stearate, not only soften tissue, especially for the ratio 
Than the tough, hard tissue, and because acid is a weak acid, mordant effect on nuclei, nuclear cytoplasm ratio distinctive, but easy to take off pieces; same 
When loose tissue easily scattered pieces). Conditional paraffin lid can be opened first day, so xylene evaporate. Used paraffin wax dip 
If the impurity filter as possible, to prevent attached to the tissue, causing the knife blade is damaged, broken or sliced and increased scratches. 
     6. Embedding 
     Embedding medium to support the organization with the process called embedding. The most commonly used is paraffin embedding. One key embedded flat, and second position. 
When asked embedding should be used tweezers, gently press arched portion of tissue, making it flat on the bottom, usually the largest organization of bread buried. Bag 
Wall, the lumen organizations should vertically embedded. Small multiple satellites organization should be put together, and to ensure that on a plane. I repair to both sides of the wax 
  (Called sides, means the slices with knife sides perpendicular). It should also pay attention to whether the embedded thread, paper wadding, if any, must be removed. 
Gastric biopsy specimens, not by the general rule take maximum embedding face, narrow face should be taken to put up embedded. Embedded in the wax should filter, leaving 
Paraffin impurities, likely to cause pollution or damage to the blade. The melting point of the wax should be between 56 ~ 58 ℃ choice, do not advocate the use of soft wax in the winter. 
Because the wax with a soft wax-embedded blocks, in the summer, will stick together, not conducive to the preservation of information, and even in winter, with a hard wax-embedded 
The wax block with a soft wax than wax-embedded blocks better cut. 
     7 Brothers 
     To cut a slice is good, we must first have a sharp knife. Brothers are engaged in a basic technical sections of technical personnel, 
Knife mill is directly related to the quality of the slice. Sharpening the way everyone is different, but require force uniform, so that the whole knife and whetstone 
Surface contact points should be in the hands of the force on the blade, rather than on the back of a knife. Application of a knife mill once every 10 to 15 minutes only, 
Over a long period of sharpening, easy to grind the blade has worn away, check the knife is sharp, knife hand to touch the test method is not very 
Science, not only can not prove that the knife really sharp, and easy to damage the blade, the easiest way is to take a well after each grinding wax 
Block trial cut it. After each wear a good knife, a whetstone should be covered with a lid to prevent dust off the grindstone, with gray grindstone sharpening, 
Slicers will produce many small gaps. Now many units are bought Grinder, Grinder used to open the blade and grinding gap really good, 
However, due to the angle and time is not easy to accurately grasp the knife, so the effect is not ideal. Based on our experience, the real blade is difficult to use machine 
Grinding out. Disposable blade largely solved this problem. If using disposable blades, blade must be replaced. A knife-edge 
Cut a small sample should not exceed 20 to 25 wax blocks, cut into large specimens should not exceed 10 to 15 wax blocks. 
8 slices 
     The first step must be rough-cut slices. The thickness of the rough cut of about 50 to 100 microns, hard tissue or smaller organizations should 
Then thin, rough-cut cause tissue all exposed only after fine cut. Fine cut to the tissue surface uniform, no white point, then cut 
Wax into the warm water. Slice requirements force uniform, soft, shaking speed too fast or too slow is not easy. Too quickly can cause uneven slice thickness, machine 
WEAR; too slow will slice thickening. Slice thickness is generally 3 to 5 microns. Slice request is complete, a thin and uniform. The cut sheet membrane 
Its size and shape should be consistent with tissue slice incomplete, and often will be an important omission lesions, leading to misdiagnosis. In the slice put wax block 
, Should pay attention to the direction of tissue embedding, hierarchical organization, fiber, muscle, etc. to be parallel with the knife, cut the part should be more difficult to put 
In the above, such as the skin, the epidermis, the coated lumps, gastrointestinal serosa, etc., can reduce the breakage of the tissue. Operation slicer 
Should be firmly and evenly to avoid excessive force. On decalcified tissue, bone marrow, and known calcified tissue should be used in a fixed blade to cut 
Less likelihood of other parts of the gap occurs. Exhibition of films using a brush pen to prevent the wire into the incision, because each cut into a wire pen, it will 
Add a gap. Slice thickness of 4 to 6 microns, some people think: Just slicer scale marked a few microns, cut out of the film 
Is a few microns. In fact, when the knife is not sharp, or when speed uneven slice or cut slower, the cut of the film will be thicker, 
Only in a very sharp knife, slice uniform situation in order to truly ensure its thickness. This is the reason for the problems encountered when sliced 
And methods that might be addressed: (1) organizational brittle hair: usually dehydrated, transparent, wax dipped a long time, temperature and texture of the organization itself 
Also relevant when sliced, side trimming wax blow to the mouth, it may be better. (2) slices rolled up, probably not sharp knife or blade in 
The other side, or the angle is too large knife, sliced thick and so on. (3) wax bent: It may be uneven blade, slicing knife unground straight, slicing knife and wax 
Blocks are not parallel. (4) transparent, wax dipped a long time, temperature, and texture is also related with the organization itself (5) uneven thickness: It may be a knife, 
Knife and wax block unclamped, the organization is too hard, or too forward slicing machine spindle, or slicer worn. (6) sliced cracks: It may be a knife 
There are gaps, there are impurities in the paraffin, calcification, bone chip or cable knot within the organization, there may be tissue fibers. (7) is not contiguous slices, cut 
Less than tablets, sliced very thick, or sliced white wax block, retraction: poor tissue dehydration. Remedy: to first dissolve the wax block, remove 
Organization, into the heated water bath of acetone (80 ℃), 30 ~ 60 minutes, then dip transparent wax-embedded sections, maybe a bit better. 
     9 exhibition piece and fishing tablets 
     Exhibition pieces temperature should be between 42 ℃ to 47 ℃, which is mainly related to the melting point of the wax and embedding, the water temperature is too high, can cause tissue cells spread, 
The water temperature is too low, not sliced smoothing wrinkles. Embedded in the wax, if any, xylene, paraffin wax can also cause dissolution phenomenon. And with a disposable blade cut 
Film, a touch of hot water, wrinkles on the film can no longer expand, there are two ways to solve this: First, you can cut in the side of the slice 
Side to wax blowing, this film will be very smooth, put hot water will naturally expand, more convenient, but this film 
Slightly thicker; Second, After cutting the film, the first slice on the 30% alcohol solution, so that the tension automatically wrinkles alcohol Exhibition 
Open, and then moved to a hot slice, so that the film will be thinner; such as alcohol concentration is too high, easily lead sliced broken, cracks appeared, like 
Adipose like cell adhesion smaller organizations touched alcohol will be spread out, it can not use this method. Finally fishing films slides should be clean, to 
Select those full, no wrinkles slice, paste in the slide in the middle 1/3 and 1/3. 
     10. Grilled slices and dewaxing 
     Grilled slices, generally 60 ℃ temperature inside the roast slices of about 0.5 to 1 hour, the temperature is too high, can cause cell contraction sliced; time is too short (less 
In 20 minutes), resulting in off-chip easily. Dewaxing is also important, if not clean dewaxing, sliced difficult uneven coloring or colored, so 
Dewaxed with xylene to be changed frequently, usually with three xylene per 500ml of liquid, 500 post-treatment biopsy once a suitable replacement. 
When the temperature is below 18 ℃, must be sliced out from the oven immediately placed xylene dewaxing, or xylene into the temperature inside to warm up (37 
℃ or so) dewaxing, shall not exceed 60 ℃. The high concentration of alcohol and low concentration of alcohol wash xylene to water. 
     11 dyeing 
Hematoxylin according to the old time, temperature, slice depending on the type of dye, usually 5-15 minutes. Slightly washed with water, salt 
Acid alcohol differentiation, differentiation must be controlled with a microscope. After differentiation washing, use warm water or water of blue and thoroughly rinsed with water (water 
Water for 5 minutes), the residual hydrochloric acid cause the slice to prevent discoloration. We do not advocate the use of an alkaline solution (release ammonia, soapy water, etc.) to promote the blue, 
Although the blue effect is very good, but the results of practice, the blue chips with an alkaline solution to promote the long-term preservation is not easy to fade. Finally into 
Eosin stained (5-20 seconds). The key lies in HE staining moderate depth, contrast, generally have experienced, the naked eye can judge, but even 
And there will be mistakes, so the microscope control is the safest method. The key step is to hematoxylin stained blue after a good differentiation of 
Process, at the end of the blue, should be observed under the microscope nucleus.The is appropriate, nuclear structure is clear, if there is residual cytoplasm 
The hematoxylin and so on. Ideal stained sections under the microscope should be: the nucleus and cytoplasm should be matched blue and red, bright beautiful; karyoplasmic contrast 
Obviously, the nuclear membrane and nuclear chromatin particles visible. 
12. Dehydration and Fengpian 
Sliced dehydrated alcohol concentration should be low to high concentrations of alcohol, 80% alcohol, a 95% alcohol 2, pure alcohol 2, (1-butanol 
Road), xylene 2. Low concentrations of alcohol higher than the alcohol concentration is more easily dehydrated, but also tends to fade eosin, the dehydration time may be shorter
Point, 10 minutes 3-5 minutes of pure alcohol per channel per channel. To increase the blending of alcohol and xylene, p-xylene can be added in front of a 
Road butanol; carbolic acid - xylene solution also has this effect, but if not used when carbolic acid washed, sliced can cause fading, is not conducive to slice Jiucun, 
It does not make recommendations. Slice after xylene, with neutral gum cementing. In order to increase the transparency of the slice, prevent cell shrinkage, cracking or 
Sliced black crystal-like dots appear. So we have a wet seal slice provisions shall not be dried with a hair dryer or oven dried and mounted. 
In the southern winter, Meiyu season, were mounted to prevent the nose and mouth when exhaled gases come into contact with slice; the weather is humid days, not one 
Times to remove multiple sections to be sealed to avoi