简述一下石蜡切片机和冰冻切片的原理及其应用

A paraffin section technique

Paraffin section method, including materials, fixed, washing and dehydration, transparent, paraffin, embedding, sectioning and stick piece, dewaxing and staining, dehydration, transparent, sealing pieces and steps. General organization made from materials fixed to the sealing piece glass samples need a few days, but the long-term preservation of specimens can be used, for permanent microscope slides specimens.

General steps, as you know, not much said, the value that a few pay attention to the place

1. Under the premise of fixed organization should be in convenient as small as possible, fixed fluid source according to the organization to choose the right. The amount of fixed liquid usually for material

About 20 times, fixed time according to the size of the block material and loose degree and the penetration rate of the stationary liquid, can be from 1 hours to several days, usually a few hours to 24 hours. Pure alcohol can be fixed glycogen and can dissolve fat, formaldehyde can fixed organization, but dissolved glycogen and pigment; A single fixed liquid cannot be fixed ingredients in the cells; Mixed fixed liquid can be complementary, the commonly used mixed liquid have Bouin's liquid and Zenker's fluid, FAA, Carnoy's liquid, SuSa fluid (see formula related technical books).

2. To prevent dehydration dehydration excessive primers tissue atrophy. If not timely to all levels of dehydration, the material can be preserved in 70% alcohol, because of high levels of alcohol easy shrink organization hardening, shoulds not be too long.

3. The dipping time is according to the organization of transparent agent material block size, and belongs to the lumen or parenchymal organs. If the transparent time is too short, the transparent incomplete paraffin difficult to immersed in organization; Transparent time is too long, the organization hardening brittle, easy cut into slices, complete the longest one hour.

4. Slice thickness and slice too thick and thin all can influence the next experiment, a thin strips with roast bad to do, may be dyed the result is not all; Dyeing is ugly, too thick, it may be or appear a very trouble.

Antigen repair, paraffin section of the biggest downside is to repair antigen, a repair is not good, the best immune dyeing is not pretty, this is a lot of people choose the biggest cause of frozen section, because there are may be one of your antigen repair optimization will take you a lot of time here.

2. Paraffin section technology advantages and disadvantages

Histological routine paraffin section (paraffin section) production technology of the most widely used method. Paraffin section is not only used for observing the morphology of normal tissue structures, and pathology and forensic science disciplines in the study, observation and judgment organization cell morphological changes of the main method, but also has been quite widely used in many other fields of research. Teaching, then most of our observation biopsy specimens of paraffin section method. And on the immunohistochemical this block. Frozen section procedure is simple, the production process of antigen activity lost less, but less tissue cell morphology; Paraffin section of the step is various, different antigen activity to reduce production, but tissue cells form is clear, is one of the routine immunohistochemical preparation section method. General paraffin embedding tissue section within the cytoplasm or used in the detection of nuclear antigen, for surface antigen staining. Cells but, paraffin embedding tissue DNA content analysis is paraffin embedding tissue section combined with flow cytometry using DNA

Content and times body analysis, this combination is the flow cytometry instrument in clinical application, especially in cancer research opens up a new research approach. Has been measured cell size, volume, DNA content, DNA synthesis rate, RNA content, surface antigen, chromosome, etc. Because of sample preparation techniques, in the past, many data flow analysis is limited to use fresh tissue samples, and now can analyze paraffin embedding tissue section dispersible cell suspension technique for the detection of DNA content, from biopsy can obtain a sufficient number of single cells, and the efficacy of a single cell with fresh tissue on the morphological and DNA content formula figure are very similar.

3. Frozen section technique

Frozen section is a low temperature fast cooling conditions to enable an organization to a certain hardness and then slice method. Because of its production process a paraffin section of the quick, convenient, and more applied in the operation of fast pathologic diagnosis. Frozen section of the sort is more, there are low temperature cryostat frozen section method, the carbon dioxide frozen section method, methanol cycle refrigeration and frozen section method, etc. With these methods, the changes of The Times, the development of science and technology, many years ago is considered very important technologies, also be phased out now. Of course some technologies, such as low temperature cryostat frozen section method, is popular.

General steps as you all know, also not much said

1. Materials: organization fresh quench the sooner, the better, as far as possible, to avoid ice crystals. The ice crystals will produce irreparable damage to cells, directly affect the immunohistochemical results. So, generally USES the organization suspended in liquid nitrogen will organize 10 to 20 seconds after critical surface quenching in liquid nitrogen.

2. The temperature of the cryostat

Generally in frozen after 10 minutes, to cut again closer to cold chamber temperature, normal tissues in 15 ~ 20 ℃ slicing most likely to succeed. Each organization has its appropriate temperature, the temperature of the knife and sliced cold chamber temperature.

3. Slice

Organize fresh because of the frozen section and quench the embedding in OCT, organization itself actually no penetration into the embedding medium, so the organization itself is soft. The technical requirements of slice is higher, sharp blade, blade Angle is good, the tissue temperature and the single chip temperature and body temperature difference appropriate; Asked slicing feed

Speed and strength should be right to cut out a satisfying film. And the leading edge of the volume resistance plate and knife surface must have enough distance, smooth slicing through. Rolled plate slightly above the highest point on the surface of the knife, resistance to can adjust the Angle of knife to the organization by experience.

4. The advantages and disadvantages of frozen section

(a) advantages:

1. Simple, can don't need to tissue fixation, dehydration, transparent, embedding and other procedures can be cut,

To reduce the intermediate links.

2. Quick, short.

3. Organizational change is not big.

4. Can save fat, lipid composition, etc.

5. To compare intact save various antigen activity and enzymes, especially for that

Some of organic solvent or the temperature of the thermal resistance is poorer membrane surface antigen and hydrolytic enzyme preservation is better.

(2) disadvantages:

1. It is not easy to do serial section.

2. Cut out organizations cannot too big, too much is not easy to freeze or organization

Uneven tissue freezing, sectioning and staining effect.

3. It's not easy to make thinner slices.

4. Tissue piece of easy to produce water of crystallization in the process of freezing and affect the cell morphology and antigen localization, and the structure of the organization than the paraffin section is clear.

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