First, in order to meet the requirements of immunohistochemistry technology, the tissue is fixed as fresh as possible.

In the judgment of the final result of immunohistochemistry, a uniform non-specific staining phenomenon is often seen, which is considered to be a pseudo-non-specific staining by various studies. Because the antigen contained in the tumor tissue is more prone to diffusion and diffusion, the unrestricted growth and growth of the tumor cells cause the blood supply in the middle part of the tumor to be difficult, resulting in ischemic necrosis. The antigen in the necrotic cells can be affected by the body. Evenly interspersed between the cells and the interstitial cells, this is a way of antigen dispersion. Another way to disperse the antigen is because the tissue is not fixed in time. The isolated tissue is not fixed in time, the tissue will be autolyzed, and the antigen will spread. This is a very common common sense, but it is extremely difficult to do well. It takes a period of time for the specimen to be surgically removed to immerse in the fixative, during which time some of the antigen can spread. Although the fixed solution has been immersed, the specimen is large, and the amount of the fixative is insufficient. Of course, since the penetration of the fixative takes time, when it penetrates into the tissue, the middle cell has changed, and the antigen also spreads. This phenomenon is more obvious in organs that produce more enzymes, such as gastric cancer. When the specimen is removed, the specimen is larger. Although the fixative has been placed during the operating room, the fixative should pass through the muscle layer to reach the gastric mucosa. It takes several hours for the tissue to change when the fixative is active. Therefore, this meets the requirements of immunohistochemical staining, and the isolated tissue should be fixed as soon as possible. If necessary, it should be cut open, taken early, and fixed early.

Second, tissue dehydration must be thoroughly clean

The tissue block should not be too thick and too thick to complete the dehydration process. If the material is too thick and the dehydration is incomplete in a short period of time, it will cause a series of problems, such as incomplete waxing, poor completion of the slice, and incomplete cutting. Due to congenital deficiency, the subsequent slicing of the slice stains, resulting in the failure of dyeing, or repeated operations, resulting in waste of human and material resources, resulting in delays in the publication of pathological reports. Therefore, the requirement for the material is that in addition to the requirement to be artistic, that is, the leveling, the appearance is good, and the requirements are moderate.

Third, the slice must be complete, uniform, flat, no lazy

For the sections used for immunohistochemical staining, the quality of the sections is high, the sections must be intact, flat, no bubbles, no folds, which is advantageous for the washing during dyeing, which is conducive to the firm attachment of the slices. If the slice is not flat, after immunohistochemical staining, uneven dyeing may occur, and the color is different in depth and uneven. If the slice has a bubble slice at the time of baking, since the rupture of the bubble affects the tissue surrounding the bubble, a dyeing of different shades can be observed around it. If the slice has a Zou fold, after immunohistochemical staining, there are different shades of color in the place where the Zou fold, which is a false positive, easy to lead to confusion.

Fourth, the attached patch must be strong, you must use a suitable adhesive.

The preparatory work before immunohistochemical staining is that new slides must be processed. The surface of the new slides looks very clean. Some people think that it is suitable for use without any treatment. This is a kind of Wrong idea. The new factory-made slides are covered with a layer of grease-like substance. If left untreated, it is extremely unfavorable for the attachment of the slices. Our approach is: new slides, soaked in glass cleaning solution for 4 hours or even overnight, then taken out, thoroughly rinsed with tap water, immersed in alcohol for more than 2 hours, removed and dried for use or dried. The slides were then immersed in a dilution of 3-Aminopropyl triethoxy-silane (1:50 diluted with acetone or absolute ethanol) for 10 minutes, after which no Wash the water ethanol twice and use it after drying.

Fifth, the slices must be baked and attached firmly, not only to withstand the high temperature of antigen repair, but not easy to remove the film, and not destroy the antigen.

Slices applied to immunohistochemical staining. Because the whole process needs several stages of treatment, such as antigen retrieval fluid boiling and needs to last for ten minutes, PBS is repeatedly washed, and some even incubate in a refrigerator at 4 ° C for more than ten hours. Therefore, the quality of the slices is very high, especially the degree of attachment of the slices. In the past, it was suggested that the slice should be baked at 60 ° C for 30-60 minutes. The result is that the slice is severe and the slice looseness is 100%. It is only appropriate for the slice to be baked for a long time, neither to release the film and to suit various requirements, and to not destroy the antigen. Several sets of experiments [] Diagnostics P173 believes that the slices are best baked in a constant temperature drying oven at 60 ° C for 2-5 hours. This is because: the antigen can tolerate such temperature, the paraffin wax generally used in pathology has a melting point of about 60 ° C. When the tissue is immersed in wax, the temperature in the waxing tank is generally adjusted to about 65 ° C, and the tissue is at such a temperature. It should be soaked for more than 2 hours to achieve thorough waxing. The antigen in the tissue has been tested at a temperature of 65 ° C, and the preserved antigen has been heat resistant. In addition, the slices baked after the above time are firmly attached, the antigen is preserved, and the dyeing success rate is 100%, which ensures the timeliness and accuracy of the pathological diagnosis.

It is important to note that whether it is applied to a diagnostic slice or a study slice, it should be stained as soon as possible after baking. If the slice is cut, it will not be dyed, stored at room temperature or in the working refrigerator. The antigen in the slice will gradually disappear with time, and even a false negative will occur. In principle, fresh slices, that is, row dyeing, how many pieces are cut, so as to preserve as much antigen as possible.

Six, slice dewaxing must be clean, otherwise it will affect the final results of immunohistochemical staining.

Wax is insoluble in water and does not fuse with other clinically used antibodies. It can only be processed by special reagents for a sufficient time to remove it. If the dewaxing is not clean, a little wax remains on the slice, which will cause many disadvantages, such as uneven dyeing, looming time when the positive substance is present, true and false, and background dyeing. In order to solve the above problems, the chips must be thoroughly dewaxed before dyeing. The reagents currently used for dewaxing are mainly xylene, because of its strong dewaxing power and short dewaxing time. More popular with users. The time for dewaxing is different depending on the season, the room temperature and the freshness of the reagents. If the room temperature is higher in summer and the dewaxing reagent is fresh, the dewaxing time does not need a lot, and 3-5 minutes is enough. If the room temperature is lower in winter and the dewaxing reagent is older, the dewaxing time needs to be extended, 10-20 minutes or longer. In short, the operation should be based on different seasons, different room temperature, different reagents, dewaxing time, in principle, thoroughly, clean, completely remove the wax on the slice. In order to do this, the warming of the slices prior to dewaxing will help to accomplish the above requirements. For example: sliced ​​on the same day, dyed after 2 hours of barbecue, and sliced ​​with temperature for dewaxing. This will speed up the process of dewaxing. If the slice is cut in advance, the slice must be warmed before dyeing. 10-20 minutes, then dewaxing, so that the dewaxing speed is faster, the effect is more beautiful.

7. The activity of endogenous peroxidase must be completely inhibited in order to reduce the staining of the background.

There are many endogenous peroxidases in various tissues, especially in red blood cells, neutrophils, monocyte eosinophils, hemorrhagic tissue necrotic tissues, and the like. If the cells and tissues containing this enzyme are not treated and inhibited prior to staining, they will catalyze the same substrate as HRP in the development of DAB substrate, allowing it to develop color and produce positive and negative substances. The same yellow-brown, causing confusion. Until now, endogenous peroxidase must be inhibited before immunohistochemical staining. Of course, it is impossible to completely eliminate them, because the inhibition is too strong, and the antigen will have the same inhibition. When hydrogen inhibits endogenous peroxidase, it should also be noted that the inhibition of antigen protection can only show a yellowish color after most of them are developed in DAB, which is enough to be true with positive the difference. The inhibitor is usually 0.3% H2O2, which can also be called 1% H2O2, because the concentration of commercially available H2O2 is 30%, and the net content is 0.3%, and if it is used, it is 1%. .

Regarding the use of H2O2, some use is a simple H2O2 dilution solution, and some use a H2O2 methanol mixture. According to the experiment, it is considered that H2O2 methanol is more suitable for most cases, because in addition to H2O2, methanol has a passivation effect on various enzymes, so the double action of H2O2 methanol is better. Chen Shangping believes that in most cases, satisfactory results have been obtained with methanol H2O2.

8. Proper and reasonable use of blocking reagents

In order to reduce the non-specific staining produced by the background, in the process of immunohistochemical staining, non-immune animal serum is often added to reduce background staining before incubation with primary antibody. Chen Shangji et al believe that antibodies are highly charged molecules, possibly No specific binding (such as collagen) to tissue components with corresponding charges. Due to this non-specific binding, it will result in partial localization of the label (conjugate) and false positive staining of collagen. To be treated first with an irrelevant antibody may reduce the specific binding of the labeled antibody.

In the past, there were many uses of serum, such as: calf serum, horse serum, goat serum, sheep serum, rabbit serum, and there are many kinds of concentrations, such as: 1%. 2%. or 1:5, 1:10, and 1. :20.

Appropriate antibodies must be selected and properly preserved and formulated.

Nine, choose the right antibody

To date, there are dozens of commonly used antibodies in clinical practice, and in this case, they can be divided into two types.

Concentrated antibody

According to the situation used in recent years, it has many advantages, but there are also many shortcomings:

1 can be used as a standing antibody for the detection of various diseases. In the routine biopsy diagnosis of each unit, there are common cases and rare cases, especially the latter, sometimes only one case is encountered, which puts higher requirements on the application and standby of antibodies, such as In addition to commonly used antibodies, it is necessary to reserve antibodies from rare cases. This will solve the clinical diagnosis problem, such as temporary purchase, it will take a long time, which will delay the diagnosis. Practice has proved that concentrated antibodies can be used as a standing detection antibody. After purchasing the antibody, according to the number of test cases, under sterile conditions, the antibody is divided into small packages, then sealed with a wax film, stored in a low temperature refrigerator at -30 ° C, and taken out at the time of use. It is allowed to rise to room temperature and then diluted with PBS. This antibody can be stored for about 3 years.

2 low cost, the price is cheaper.

Compared with the ready-to-use antibody, the price is much cheaper. For example, in a company's 2002-2003 CK, the concentrated antibody is 0.1ml, the price is 260 yuan, and the antibody can be diluted to 5000ml, each piece. The required antibody for sectioning is 50 ul, which can be labeled in 100 cases, and the antibody required for each antibody is 2.6 yuan, while the ready-to-use antibody is 1.5 ml, the price is 150 yuan, and 30 cases can be labeled, and each antibody requires 5 yuan.

3 need to dilute the antibody, the procedure is more complicated. For newly purchased concentrated antibodies, they must be diluted into working fluids before use. For antibodies that have never been used, the actual dilution must be tested. Because of various antibodies, manufacturers have done the corresponding The dilution test, but this is the approximate dilution. To make the antibody really suitable for the dilution of the unit, it is necessary to experiment on the dilution of the antibody, such as 1:100.1:75.1:50.1:25, etc., if the result is The best result is 1:50, which is the optimal dilution point. If the best dilution point is not found within this range, continue the experiment until the optimum dilution point is found.

4 need to configure each type of micro-sampler to facilitate the measurement of the exact amount of antibody.

5 need to have a certain level of English, because concentrated antibodies are mostly imported reagents, their use of books are mainly in English, which involves the source of antibodies, adapt to what tissue or cells can react to dilution.

2. Ready-to-use antibodies.

In recent years, the application of immunohistochemical technology, the application of ready-to-use antibodies has been more and more popular. According to the use in recent years, it has many advantages and disadvantages:

1 easy to use. For beginners, it is the best antibody. No matter what kind of cases and slices, as long as there are antibodies, you don't need to explore the dilution yourself. It is very inconvenient to pick up a drop of the reagent bottle.

2 For those who do not have or do not understand immunohistochemistry, as long as they are used according to the instructions, the ideal results can be obtained.

3 do not need to configure a variety of expensive micro samplers. Today's micro-sampler, such as imported goods, you up to more than two thousand yuan. The ready-to-use antibody does not need to be diluted, ready to use, without the need for the rest of the equipment.

4 is especially suitable for the base unit. At the grassroots level, immunohistochemistry can be carried out without much investment, which is extremely beneficial for improving the accuracy of diagnosis.

5 price is more expensive than concentrated antibodies.

6 Ready-to-use antibodies are not available as a standing stock antibody. Ready-to-use antibodies are generally valid for one year. If the unit is used in a large amount, for a package of 3 ml of the antibody, several times a year is needed, which does not cause waste for the antibody. However, if it is a small unit, there are fewer cases marked in a year. When purchasing antibodies, try to choose small-package antibodies, such as 1.5ml. If you encounter a rare case, sometimes you can't mark a few cases in a year, so you should use concentrated. Ready-to-use antibodies are valid for one year, but the actual period of use of the antibody is not likely to be one year, because the antibody needs a certain amount of time from the manufacturer to the seller. If you are lucky, you buy it. The antibody obtained is just in the hands of the seller, so the use time may be longer, but if it is stuck in the hands of the seller for a while, the effective use period of the antibody may not be too long, five months , six months or three months. If you cannot use it within the valid time. It may be more than some time. If the time is too long, it should be discarded because it will affect the quality of the mark. Some people complain that the antibody they just bought is very good, and then it is getting worse. This is because the activity of the antibody gradually loses its biological activity over time, resulting in a decrease in the quality of the label.

(2) Indications for antibodies.

Among the many antibodies, they are divided into two categories according to their actual use range:

1. Suitable for frozen sectioning, smear, cell culture tablets.

2. Suitable for paraffin sectioning, frozen sectioning, smear and cell culture tablets.

(3) Select a suitable monoclonal or polyclonal antibody.

In addition to the above two classes of antibodies, there are also monoclonal and polyclonal points from the height of their cloning.

1. Monoclonal antibodies, most of the antibodies commonly used in clinical practice are monoclonal antibodies. This is due to the continuous improvement of biotechnology. In the early days, antibodies applied to the clinic were mostly polyclonal antibodies.

2. Polyclonal antibodies, among the clinically applied antibodies, a small number of antibodies are polyclonal antibodies.

(4) Addition of antibodies.

This seems to be a very simple problem. If it is not handled well, it can lead to different results. If an automatic immunohistochemical staining instrument is applied, the program can be input. If it is manually operated, the following problems must be noted:

1. When the PBS is rinsed, add an antibody such as primary antibody, secondary antibody or secondary antibody. The excess PBS remaining on the slice must be dried, but the slice can not be dried up, and the slice can be dried, often resulting in non-specific staining. Excessive PBS retention on the slice will dilute the added antibody and affect the concentration of the added antibody. It will also affect the final results, such as false negatives.

2. The added antibody is preferably one drop.

After drying the excess PBS around the tissue block, the slice maintains a certain degree of wetness. At this time, it is best to add another antibody. It is better to add a drop of 50 ml to the antibody. After the addition, gently cover the slice slightly. On the top, the final result is stable and balanced, and there is no reaction in some places, and no reaction in some places.

3. If the slice is not rubbed, it will affect the quality of the added antibody. The slice is not rinsed out. It is not wiped. When the antibody is added, it can be shrunk to the side of the slice. At this time, in order to completely cover the entire antibody. If you slice it, you will add the antibody. The result is that the antibody is still sliding to one side, or it is completely exposed. This not only fails to achieve the purpose, but also wastes the antibody. The correct way is to thoroughly rinse with PBS, dry the slice and dry it. Slice the surrounding PBS, and then add a drop of antibody and gently shake it.

(5) Select the appropriate incubation time.

The incubation time of the first antibody has a wider range of use and is applied to the incubation time of the clinical detection antibody. Generally, incubation at room temperature for 30-60 minutes, 120 minutes, for the study of antibody incubation time, can also be according to the above time, but it has also been proposed to incubate overnight in a refrigerator at 4 ° C, according to our experience is generally not advocated at 4 ° C Overnight in the refrigerator due to:

1. Long-term incubation can cause some non-specific substances to precipitate or be adsorbed, resulting in background staining.

2. Currently sold antibodies have high purity and specificity, and do not require long-term incubation, and can also be well combined.

3. Long-term incubation is more likely to cause the shedding of the slices, resulting in the failure of staining.

4. Long-term incubation is not conducive to the issuance of clinical pathology reports.

Ten, the selection of antibodies must be correct, otherwise it can cause false negative phenomenon.

There are many immunohistochemical staining methods, such as ABC method, SP method and EV two-step method. If you use SP method or ABC method, you must carefully select the antibody to be conjugated. The first antibody has a single gram. For polyclonal antibodies, the antibodies to be ligated should be based on the selected antibody. For example, the primary antibody is a monoclonal mouse anti-human ** antibody, and the antibody to be ligated should also be selected to be anti-mouse IgG so that it can be ligated. (Currently, manufacturers have produced mixed antibodies, which are suitable for both monoclonal antibodies and polyclonal antibodies. Users don't have to worry about connecting, they can be used casually, but if you don't order a hybrid kit, Must pay attention to the model, making it suitable for the selected antibody.) Incubation time can be 10 minutes, 20 minutes or 30 minutes, usually at room temperature, if the room temperature is below 20 ° C, it can be incubated at 37 ° C In the box.

11. Use of the complex and determination of the incubation time.

Various methods have their own complexes, such as the ABC method, the complex is a complex of avidin and biotin, and then the HRP, SP method, (LsAB method) complex is a streptomycin protein complex, The composite with HRP and EV two-step method is a composite of secondary and tertiary antibodies, and then carries HRP. In addition, depending on the hydrolysis substrate, different colors can be produced, for example : An enzyme with HRP. The hydrolyzed substrate is generally DAB producing a yellow-brown, AP-bearing enzyme. The hydrolyzed substrate is generally AEC producing red.

Twelve, PBS rinse

The immunohistochemical staining process, in addition to the previous treatment and the addition of antibodies, was spent in rinsing and washing in PBS, and the correctness of PBS washing was also the key to the final result.

1. Wash separately to prevent contamination from cross-reaction.

There are many cases of clinical tests every day, and there are many types of antibodies and items to be used. If they are washed in a tank after the primary antibody is added, this will cause cross-contamination and affect the final result. The correct approach is to rinse separately, and the rinsed PBS is disposable, ensuring that the slices do not have the opportunity to cross-contamination.

2. Gentle rinse to prevent the slices from falling off.

Rinse the sections, remove the sections, rinse the PBS gently from above, let the PBS flow down from top to bottom, do not pick up the slices and align the PBS with the slices, so that the PBS has a certain impact and it is easy to slice. Looseness in the periphery causes the slices to fall off.

3. Flush time is sufficient to thoroughly wash away the combined material.

Flushing the slice has been proposed to use the micro-oscillator to irritate and shake off the unbound various materials so that it does not produce a background. This is a waste of time, and the second is easy to cause the slice to fall off. It is considered that the rinsing of the slice is carried out by gently rinsing the slice with a small beaker, and the unbound material can be thoroughly rinsed without additional conditions. The rinsing is better than the PBS, and it is enough for about 2 minutes. .

4. The use and requirements of PBS pH and ionic strength.

Liu Yanfang pointed out that neutral and weak sputum conditions (PH7-8) are conducive to the formation of immune complexes, while acidic conditions are favorable for decomposition; low ionic strength is beneficial to the formation of immune complexes, while high ionic strength is beneficial for decomposition. . [] Immunohistochemical P56 The pH of PBS we currently use is 0.01M at 7.4-7.6. According to the use of this room for more than ten years, it is considered that the solution is cheap and suitable for use.

5. Preparation and use of commonly used reagents.

In the immunohistochemical staining process, the most used reagent is the buffer, because the whole process of staining, the addition, replacement of the antibody, the rinsing of the slice, and the preparation of the DAB are inseparable from the buffer. It can be seen that the buffer plays a key role in the whole staining, and the peracid or alkalinity of the buffer will affect the staining result. The following will introduce the relevant aspects:

(1) Preparation of buffer

1. Phosphate buffer solution (PBS)

Liquid I: 0.2M Na H2PO4 stock solution (pH 7.6)

Take a 500 ml volumetric flask, weigh 15.60 g of NaH2PO4.12H2O into a bottle, add a small amount of distilled water and shake vigorously until dissolved. Add distilled water to make up 500 ml. Store in 4 refrigerators, things to be aware of when preparing:

1 In the preparation, the amount of NaH2PO4 should be determined first, because the reagents have many kinds of forms, and the water molecules they contain have different molecular weights, so they are used in different amounts. For example, if NaH2PO4 contains a single water molecule, 13.80 g should be weighed when preparing, and when it contains 2 water molecules, 15.60 g should be weighed.

2 When preparing, choose distilled water according to requirements. Because there are several kinds of distilled water, single distilled water, double distilled water, glass distilled water, deionized water. If not specified, it can be prepared by using distilled water.

3 Buffered bottles must be washed with glass dipping solution to prevent contamination, resulting in the growth of snow bacteria in the solution.

Liquid II: 0.2M NaHPO4 stock solution (HP7.6)

Take a 500ml volumetric flask, weigh 17.91g NaHPO4.12H2O, pour it into the bottle, stir with water, until it is completely dissolved and then make up 500ml, store in a refrigerator at 4 °C.

Precautions:

1 The reagent should also be noted to have a variety of different aqueous molecular weights.

2 The storage solution may crystallize after prolonged release. Before each use, take it back to room temperature, shake it and dissolve it in the crystal. It can also be quickly dissolved in a microwave oven before use.

Preparation of 0.01M PBS working solution:

Take a 2000ml volumetric flask, add the so-called NaCt18g, add a small amount of distilled water to dissolve it completely, then add 13ml of I solution, 87ml of II solution, add distilled water to make up 2000ml, you can use, you can use this solution It can be stored for 3-4 weeks at room temperature, but it is better to use new ones.

After the PBS working solution is prepared, the PH value should be measured. If the acid is too acidic, it can be debugged with sodium hydroxide. If the partial alkali can be debugged with HCl, the test has the following n methods:

1PH test pen test, this is a simple, easy to test method with more accurate results. After preparing the PBS, take a small cup, pour the prepared PBS, insert the test pen, open the switch, and the measured PH result will appear on the small screen. If the PH is insufficient, the small amount can be adjusted with 0.2M Na2HPO4, and a large amount of sodium hydroxide can be adjusted. If the PH is higher than 7.6, a small amount is adjusted with 0.2M NaH2PO4, and a large amount of HCL can be used to adjust.

2 Test with test paper: There are two kinds of PH test paper, one is a wide range of PH test paper, the range is 1-14, and the other is a wide range of precision test strips. However, as the PBS for rinsing the slice, the accuracy does not need to be very precise. For example, when it is equipped with PH7.6, it can be used at pH 7.5 or 7.7. The test paper is tested for pH and is determined by the color of the reaction. It is very simple and can be tested with general reagents.

3 Instrument test: For higher requirements, more accurate PH value, must be tested with the instrument.

1.Tris buffer solution (TBS)

11N HCL, concentrated HCL (double 1.19) 8.3ml, first take 50ml distilled water, add HCL and add water to 100ml.

20.5M Tris-HCL Buffer (pH 7.6)

Take 6.57 g of Tris (hydroxymethylaminomethane), add a little distilled water and stir until dissolved, then add 1N HCL 42 ml, then make 100 ml with distilled water and store in a refrigerator at 4 °C.

When used, the above solution 10 is diluted by a factor of 10, which is 0.05 M working solution.

Precautions:

1 When formulating 1N HCL, if it is formulated in a large amount, HCL can generate a lot of heat when it enters the water. The glassware used should be especially careful to prevent the glassware from bursting due to sudden heat generation.

2 When adjusting the PH value, refer to the above three methods.

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