1. Comparison between paraffin section and frozen section?

(1) Those who require frozen slices may not be able to do paraffin slices. This is the conclusion I got from a teacher today. Because the high temperature baking slices for paraffin section may destroy the antigenicity of the tissue. If the antigenicity of the tissue is more stable, it can be used for paraffin section; but if the paraffin section is required, it can be used for frozen section.

(2) The advantage of frozen section is that it can better preserve the antigen immunoactivity of the tissue, and the step of antigen repair is not required for immunohistochemistry. The disadvantage is that ice crystals are easily formed in the cell and destroy the cell structure, which may cause antigen diffusion; the thickness of the slice is thicker than paraffin, and the film is not as beautiful as paraffin. When you buy a primary antibody, the catalog reads what kind of slices to make. If it says it can only be frozen, it cannot be paraffin. If it says both, it can be done.

(3) The advantages of paraffin sections can maintain the morphological structure of tissue cells and are easy to store at room temperature. Frozen sections are more troublesome and must be stored in a low-temperature refrigerator at -80 degrees, especially for in-situ hybridization sections. To prevent RNA degradation, preservation is always important. Since paraffin sections can be cut to about 4 microns, the in situ hybridization probe easily penetrates into the tissue and is easy to succeed, and the color / morphology obtained are better than frozen sections.

2. What are the main points and skills of primary antibody selection?

(1) Selection of monoclonal and polyclonal antibodies. The specific antibody produced by a clone is called a monoclonal antibody. Monoclonal antibodies can specifically bind to a single specific antigenic determinant, just like a missile hits a target precisely. On the other hand, even if it is the same epitope, antibodies can be produced by several clones in the body, forming a hybrid of several monoclonal antibodies, called polyclonal antibodies. In the antigen-antibody reaction, monoclonal antibodies generally have strong specificity, but the affinity is relatively small, and the detection sensitivity of the antigen is relatively low; while the polyclonal antibody has a slightly weaker specificity, but the antibody has a strong affinity and high sensitivity, but is prone to non-specific staining (Can be avoided by closing etc.).

(2) Selection of application range. Some primary antibodies can only be used for Western blotting, or immunohistochemistry, immunofluorescence, immunoprecipitation, etc .; even indicate paraffin sections or frozen sections.

(3) Species reactivity. This is very important, indicating that there may be species differences in this antibody, and this antibody is suitable for detecting the antigen of which species of animal body.

(4) Species of origin, most of the rabbits are polyclonal; and the mice are mostly monoclonal, but there are other. Based on this source, select the appropriate secondary antibody.

(5) The choice of the manufacturer. For example, the antibody of Santa Cruz company is generally 1ml, the price is about 2100 yuan; while the primary antibody of BIOAY company is generally 100ul, the price is about 1600 yuan. The actual titers of the same antibody from these two manufacturers are different. I generally use the latter antibody for immunohistochemistry, but the former can do Western blotting.

3. When to use Triton-X100

(1) Triton X-100 chemical name is polyethylene glycol octyl phenyl ether, which is a detergent. In immunohistochemistry (> 10um thick sections) and immunocytochemistry, Triton X-100 is generally used as a cell permeating agent, and the membrane is perforated.

(2) Its principle of action: Triton X-100 can dissolve lipids on cell membrane, nuclear membrane, and organelle membrane to make antibodies and macromolecular structure substances enter the cytoplasm and nucleus, so it is especially recommended for cellular immunohistochemistry Use, so that the antibody can smoothly enter the cell and bind to the corresponding antigen.

(3) Triton X-100 is both a surfactant and an antioxidant.

4. What is the selection principle of blocked serum?

(1) There are remaining sites on the membrane or slice that can non-specifically adsorb antibodies, causing false positives for subsequent results!

(2) The blocking serum is generally from the same source as the secondary antibody. The animal's own antibody in the serum can bind to the site of cross-reactivity in the tissue in advance. Otherwise, if it binds to the secondary antibody in the subsequent steps, it will cause background.

(3) Calf serum, BSA, sheep serum, etc. can also be used, but they cannot be consistent with the source of the primary antibody.

5. Comparison of antibody incubation conditions?

(1) There are several kinds of primary antibody incubation temperature: 4 degrees, room temperature, 37 degrees, of which 4 degrees is the best; incubation time: this is related to temperature and antibody concentration, generally 37 degrees 1-2h, and 4 degrees overnight and from After taking out the refrigerator, reheat at 37 degrees for 45 minutes. (

2) The secondary antibody is usually room temperature or 37 degrees 30min-1h, the specific time needs to be explored.

6. Why do I need to rewarm at 37 degrees after the primary antibody is incubated at 4 degrees?

(1) On the one hand, prevent the slices from being put into PBS easily from 4 degrees to take off the slice;

(2) On the other hand, make antigen-antibody binding more stable. Generally not needed, but may be useful for weakly expressed antigens. The molecular motion is different at 4 degrees and 37 degrees. The former has a lower probability of collision and faster movement than the latter. The latter combines faster, but the sensitivity is also improved and easy Causes non-specific staining.

(3) Actually, I agree with the latter statement, because I tried to take the liver or testicle slices from 4 degrees overnight and washed them directly with PBS. Facts speak louder than words! 7. How to grasp the color development time of DAB?

(1) The color development time of DAB is not fixed, the color development time is mainly controlled by the microscope, and it can be rinsed when the light brown background appears;

(2) The DAB color development time is very short (such as a few seconds or tens of seconds) and a dark brown color appears, which may indicate that your antibody concentration is too high or the antibody incubation time is too long, you need to lower the antibody concentration or shorten your The antibody incubation time;

(3) In addition, if the background appears very deep for a short time, it may be that the blocking non-specific protein in front of you is incomplete, and the blocking time needs to be extended;

(4) The DAB coloring time is very long (such as more than ten minutes) before positive staining appears. On the one hand, it may indicate that your antibody concentration is too low or the incubation time is too short (preferably the primary antibody 4 degrees overnight); on the other hand The closure time is too long.

8. How to analyze the results of immunohistochemistry?

(1) Positive staining cell counting method. Under 40 * light microscope, randomly select 10 fields of view that are not overlapping, count positively stained cells manually or by machine, and 3 to 6 different animal tissue sections in each group, and then compare between groups.

(2) Ash density analysis method. By selecting the same area on different sections and different animal tissue sections, under the same conditions, use image j to perform gray density analysis, and then perform statistical analysis.

(3) Scoring method. Tissue sections were scored according to the degree of staining (0 ~ 3 divided into negative staining, light yellow, light brown, dark brown) and positive range (1 ~ 4 divided into 0 ~ 25%, 26 ~ 50%) , 51 ~ 75%, 76 ~ 100%), the scores can be added together, and then compared. For the above methods, please choose carefully. The prerequisite for correct results is that you have to make high-quality slices with uniform shading and a light background.

9. Under what circumstances does tissue antigen repair occur and what are the conditions for antigen repair?

(1) As some antigens in the tissue are fixed in formaldehyde or paraformaldehyde, cross-linking of proteins and blocking of aldehyde groups occur, thereby losing antigenicity. Through antigen repair, the intracellular antigenic determinants are re-exposed, and the antigen detection rate is improved.

(2) The repair methods are generally divided into three types from strong to weak, high-pressure repair, microwave repair, pancreatin repair. There are also several types of repair fluids (specific information can be found, a large number: neutral, high pH, ​​etc.).

(3) Microwave repair, we usually use 6min * 4 times, the effect is good.

10. What is the inactivation time and concentration of endogenous peroxidase?

(1) Generally, the inactivation time of 3% hydrogen peroxide is short, which can be about 10 minutes; and 0.3% hydrogen peroxide can appropriately prolong the closing time, generally 10 to 30 minutes.

(2) The configuration of hydrogen peroxide with methanol may be better than double distilled water or PBS in protecting the antigen and fixing the tissue. Hydrogen peroxide incubation time is too long to cause off-chip.

(3) Use it now and keep it in the dark at 4 degrees.

11. How to fully dewax?

(1) Wax is insoluble in water. If dewaxing is not clean, a little wax will remain on the slice, which will cause uneven staining, loosing of positives, illegibility of true and false, and increased background staining. In order to solve the above problems, the slices must be thoroughly dewaxed before staining. The reagents currently used for dewaxing are mainly xylene, because of its strong dewaxing power and short dewaxing time;

(2) The time of dewaxing depends on the season, the room temperature and the freshness of the reagent are different. If the room temperature is high in summer and the dewaxing reagent is also fresh, the dewaxing time does not need to be much, 3-5 minutes is enough. If the temperature is low in winter and the dewaxing reagent is also old, the dewaxing time needs to be extended, 10-20 minutes or longer.

(3) The slices cut on the same day are stained after baking for 2 hours. The slices are dewaxed with temperature. This will accelerate the dewaxing process. If the baked slices are cut in advance, the slices must be added before dyeing. Warm for 10-20 minutes, and then dewax, so that the speed of dewaxing is accelerated, and the effect is better. In short, the operation should be determined according to different seasons, different room temperature, different reagents. In principle, the time of dewaxing is to completely, cleanly and completely remove the wax from the slice.

12. How to minimize tissue non-specific staining?

(1) Shorten the primary antibody / secondary antibody incubation time and dilute the antibody to control. This is the most important one.

(2) Polyclonal antibodies for primary antibodies are prone to non-specific staining. It is recommended to try out monoclonal antibodies.

(3) Endogenous peroxidase and biotin are highly contained in liver, kidney and other tissues (tissues with many blood cells), and it is necessary to reduce the background staining by extending the inactivation time and increasing the concentration of the inactivator;

(4) Non-specific components are combined with antibodies, which requires increasing the blocking time of the animal immune serum derived from the secondary antibody and appropriately increasing the concentration to enhance the blocking effect;

(5) Reduce DAB incubation time or reduce DAB concentration / hydrogen peroxide concentration, etc .;

(6) Properly increase the number of PBS rinses and the immersion time, especially the immersion after the primary antibody, secondary antibody or SP incubation;

(7) Prevent dry slices during the staining of specimens, which easily enhances non-specific staining.

13, the grasp of hematoxylin counterstaining time?

(1) The time of counterstaining hematoxylin depends on the room temperature, the old and new solutions, and the location of the target antigen. It usually takes a few seconds to a few minutes. But this can be remedied if the dyeing is not ideal. That is to say, the deeper staining can make the differentiation time longer; the lighter staining can be placed in hematoxylin and then stained.

(2) Hydrochloric acid and alcohol are differentiated, and ammonia is returned to blue. The role is different. After counter-staining, the film was washed with running water, and then placed in hydrochloric acid alcohol for a few seconds (must be fast), take out the running water and wash it, then put it back in the ammonia.

(3) If the color of differentiation is too light, it can be placed in hematoxylin and stained.

14. How to choose the cleaning method, frequency and time of PBS?

(1) Flush separately to prevent contamination caused by cross-reaction. There are many clinical cases detected every day, and there are many types of antibodies and items. If the primary antibodies are added and incubated, they will be washed in a tank, which will cause cross-contamination and affect the final result. The correct approach is to rinse separately. The rinsed PBS is disposable, to ensure that there is no chance of cross-contamination of the slices.

(2) Rinse gently to prevent the slice from falling out. Rinse the slices, remove the slices, gently rinse the PBS from the top, let the PBS flow down from the top, do not pick up the slices and rinse the PBS at the slices, so that the PBS punched out has a certain impact, it is easy to make The surroundings cause looseness, causing the slices to fall off.

(3) The rinsing time must be sufficient to thoroughly remove the combined substances. Washing slices Some people proposed to use a micro-vibrator to vibrate and stain the unbound various substances so that they do not generate background. This method is a waste of time, and it is easy to cause the slice to fall off. Rinsing of slicesAccording to practice, gently rinse the slices with a small beaker to rinse out the unbound substances without additional conditions. Rinse better than slices and inject PBS for about 2 minutes. .

(4) Use and requirements of PH and ionic strength of PBS. Neutral and weak alkaline conditions (PH7-8) are conducive to the formation of immune complexes, while acidic conditions are conducive to decomposition; low ionic strength is conducive to the formation of immune complexes, and high ionic strength is conducive to decomposition.

(5) Preparation and use of common reagents. In the immunohistochemical staining process, the most used reagent is the buffer solution, because the entire staining section, antibody addition, replacement, section washing, DAB preparation are inseparable from the buffer solution. It can be seen that the buffer plays a crucial role in the entire staining. The over-acid or partial alkali of the buffer will affect the staining results.

15. What are the causes of peeling and how to prevent peeling?

(1) The quality of polylysine slides. I originally bought it, and it is said that the new brand is also good, but what it looks like. It is better to supplement the film made by the pathology teacher used in the second batch later.

(2) The tissue is not cut well. The problems of the slicing machine are, for example, the thickness of the old machine is not uniform, or the slicing technique is not good.

(3) The problem of tissues. I use a lot of cancer tissues. The more cancer tissues have necrosis, the easier it is to take off.

(4) It is not baked well, the time is short and the temperature is not enough.

(5) When the operation is too sharp, it is best not to shake or gently shake the film that is suspected of being taken off, and slowly absorb water from the edge with toilet paper.

(6) The problem of repair: when the antigen is repaired, the high-pressure time is too long, or the technique is not good when put in the repair solution of 100 degrees, and it is thrown into it with a loud sound, so it is very easy to take off the film. In addition, EDTA repair is easier to take off than citric acid, but when you want to use EDTA, there is no way to do it, only from other issues.

(7) In addition, once you see an organized float operation, you should be more cautious. Use PBS as much as possible when using PBS instead of flushing. Basically, I have paid attention to these aspects. As far as possible, I can improve it as much as possible.

16. What are the reasons for the dark background staining?

(1) The antibody concentration is too high: the primary antibody concentration is too high is one of the common reasons. The solution is to test the working concentration of each new antibody before using it, so that each antibody can be personalized and find the ideal working concentration for its own laboratory, even if it is a ready-to-use antibody, it ca n’t be simple According to the instructions for dyeing.

(2) The antibody incubation time is too long or the temperature is high: The solution is to strictly implement the operating procedures. It is best to wear a timepiece or clock with you to remind you in time to avoid prolonging the time due to forgetting. The popular two-step method (Polymer) is highly sensitive, requiring the primary antibody incubation time to be not 1 hour, but 30 minutes. Therefore, it should be adjusted according to the staining result.

(3) DAB deterioration and color development time is too long: DAB is best used now, if there is sediment, it should be filtered before use. The prepared DAB should not be stored for too long, because in the absence of enzymes, hydrogen peroxide will also release oxygen atoms and react with DAB to reduce the effectiveness of DAB. Unused DAB is stored in the refrigerator for a few days. It is not advisable to use this seemingly economical method. The color development of DAB is best monitored under a microscope, and the reaction is terminated immediately when the desired degree of staining is reached. However, when there are too many stained sheets or when using a staining machine, this seems unrealistic, but at least some new or less used antibodies should be monitored for color development to avoid excessive color development time.

(4) Dried tissues: Failure to replenish the fluid in a timely manner after the repair fluid overflows, too many stained slices, too slow movements, forgetting to drip, and dripping of the drip are all reasons that cause the tissue to dry out. The solution is to operate carefully, using DAKO pen or PAP Pen to draw a circle around the tissue, which can effectively avoid the loss of liquid and increase the operation speed.

(5) The immersion time of the slice in the buffer or repair solution is too long (more than 24 hours): the reason is not clear, but the phenomenon exists. Some laboratories like to dewax the slices to repair the day before, and add antibodies for immunohistochemical staining the next day. If the container with slices and repair solution is placed in a 4? C refrigerator overnight, there is no obvious effect on the results. At room temperature, especially in hot summer, the background coloring will appear, so it should not be stored for too long.

(6) Polyclonal antibodies with degraded primary quality and poor quality: Pay attention to the expiration date of the antibody. The expired antibody is either not colored or the background is colored. When using a newly purchased antibody, it is best to set up a positive control and compare it with the used antibody. 17. How to make ammonia water return to blue after hematoxylin counterstaining, what is the concentration, and how long? A: The blue return can be blued with alkaline solution (PBS / NA2HPO4 / light ammonia) or 45 degree warm water or cold water. Normally blue for 5 ~ 10 minutes. Some people use 50ml tap water + three or four drops of ammonium hydroxide.

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