Immunohistochemistry often encounters abnormal background coloration in addition to normal true positive signals. These abnormal colorations are referred to as " murmur " staining. There are many kinds of " murmur " dyeing, and the causes are various. For the convenience of explanation, the author summarizes the following.
1 , full coloring
Whole-slice coloring means that the entire slice is dyed with color, and the intensity of the coloration can be deep or shallow. In short, it is indistinguishable that those tissues are positive and those tissues are negative. The reasons for this phenomenon are:
( 1 ) The antibody concentration is too high: one of the common causes is that the concentration of the primary antibody is too high. The solution is to test the working concentration of each antibody before each new antibody is used, so that each antibody can be individualized to find the ideal working concentration for your laboratory, even if it is a ready-to-use antibody, it should not be simple. Dye according to the instructions.
( 2 ) The incubation time of the antibody is too long or the temperature is high: the solution is to strictly follow the operating procedures. It is best to wear the timetable or report the clock with you to remind you in time to avoid prolonged time due to forgetting. The popular two-step method ( Polymer ) is highly sensitive, and it is required that the incubation time of the primary antibody is not a conventional one hour, but 30 minutes, and therefore, it is 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 and used. DAB formulated should not be stored too long, because in the absence of the enzyme, hydrogen peroxide free oxygen atoms will react with and reduce the effectiveness of DAB the DAB, DAB unused stored in the refrigerator after a few days It is not advisable to use this seemingly economical approach. 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 tablets or when using a dyeing 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 ) Tissue drying: the liquid is not replenished in time after the overflow of the repair solution, too many stained slices, too slow movement, forgetting dripping, dripping loss, etc. are all causes of tissue drying. The solution is to carefully and carefully use the DAKO pen or PAPPen to draw circles around the tissue, which can effectively avoid liquid loss and improve the operation speed.
( 5 ) The immersion time of the slice in the buffer or repair solution is too long (more than 24 hours): the cause is not clear, but the phenomenon exists. Some laboratories like to dewax the slices to the previous day, and add antibodies for immunohistochemical staining the next day. If the container containing the sectioning and repairing solution is placed in a 4 degree refrigerator overnight, there is no significant effect on the results. Background coloring occurs at room temperature, especially in hot summers, so it is not possible to store for too long.
( 6 ) Monoclonal antibodies with poor resistance and poor quality: Pay attention to the expiration date of the antibody, and the expired antibodies either do not develop color or background coloration. When using newly purchased antibodies, it is best to set up a positive control and compare it with the used antibody.
2 , " Yin and Yang face " coloring
It means that the tissue is half-colored and half-colored without coloration, resulting in two staining results with clear or less clear boundaries. The cause is that the reagent covers only part of the tissue rather than all. If the reagent is not dispersed after the addition of the reagent, it is concentrated on part of the tissue. Usually after the addition of the reagents, look carefully, if any tissue is not completely covered by the reagents, if this is the case, it is recommended to use a toothpick instead of using a tip or reagent bottle to drain the reagent to cover the tissue. . In addition, the dyeing cassette was not flat and the slices were tilted. Although the reagents had completely covered the tissue, the reagents flowed to one side and some of the tissues were not covered by the reagents. For this kind of problem, as long as you pay attention or think of it is easy to find, it is easy to solve. Sometimes, when a DAKO (or PAP ) pen is used to draw a circle around the tissue, the line is too close or drawn onto the tissue. Due to the mechanics of the pen oil, the reagent cannot reach the tissue near the line. There are also bubbles that can cause distinct yin and yang coloration, except that the non-colored area is round. Since the gas is contained in the bubble, the reagent is pushed around, and therefore, the tissue at the center of the bubble is not colored. The solution is to use a lighter method when dropping the reagents, and use a toothpick to break when there is a bubble.
3 , slice edge coloring
Slice edge coloring is also a common phenomenon, a phenomenon known as edge effect. Causes: ( 1 ) The edge of the tissue and the slide are not firmly adhered, and the edge tissue is loosened and floated in the liquid, and it is not easy to wash the reagent under the tissue every time. Solution: Prepare high-quality film ( APES or polylysine), cut out the thinnest tissue section, no thicker than 4 microns, the pre-treatment of the tissue should be standardized, try to avoid the use of more necrotic tissue. ( 2 ) The reagent added on the slice does not cover the tissue sufficiently, and the reagent at the edge is easy to dry first, and the concentration is higher than that of the central tissue, resulting in deep staining. Solution: The reagent should cover the tissue adequately and should be 2 mm beyond the edge of the tissue . When using DAKO strokes, in order to avoid the influence of oil, the circle should be 3-4 mm from the edge of the tissue .
4 , stove-like coloring
In the section of the section, the coloring area is east and west, and it is distributed in the form of a stove. The reasons for this problem are as follows: ( 1 ) The water is not drained when the scorpion is formed, and the bubbles are formed in the local part to cause the tissue to protrude, and the reagent is not easily washed out after the dyeing. The color is too deep. The solution is that the air bubbles in the float box should be exhausted, and the hot table should not be placed flat. It should have a slope of about 45 degrees, which is conducive to water flow and evaporation. ( 2 ) necrotic tissue foci, cell destruction after tissue necrosis, enzyme release, protein free, decomposition, complex peptide chain residues (such as Fc segment) may be combined with primary or / and secondary antibodies resulting in final coloration. The solution is to avoid selecting sections with more necrotic tissue when selecting stained sections. ( 3 ) When making APES film, the concentration of the glue is too high, leaving a small white spot on the slide after drying, and white dots are colored when the color is developed. The solution is to follow the standard preparation method, namely 5% hydrochloric acid alcohol ( 5 ml hydrochloric acid + 95% alcohol 95 ml ) soak the slide for 4 hours, rinse the slide with hot water for 1 hour, wash the slide for 1 minute with distilled water , soak the glass with acetone. air-dried sheet after 5 seconds (room temperature), 2% APES (2 ml APES + 98 ml acetone) five minutes soak slides, slides over what acetone (1-2 seconds), distilled over what slides (1- 2 seconds), dry overnight at 37 degrees, store at room temperature for use. If the acetone is gradually volatilized during the tableting process, some acetone may be added as appropriate.
5 , cytoplasm staining
Cytoplasmic staining is the most deceptive coloration in all " murmur " staining. The staining area is confined within the cell, the interstitial is not stained, and it looks almost the same as the actual immune response. It is difficult to distinguish. The cytoplasm contains more protein, so many non-specific stains can appear in the cytosol in addition to interstitial. Coloring caused by this cause can be solved by serum blocking. There are also pigmentation caused by endogenous enzymes such as hemoglobin (erythrocytes), myoglobin (muscle cells), cytochromes (granulocytes, monocytes), catalase (liver, kidney), which can be used with hydrogen peroxide. Closed. Macrophages phagocytose various antigenic substances or Fc fragments and cytoplasmic staining, which is difficult to avoid, but can be recognized by morphological recognition of macrophages. The coloration of endogenous biotin is most deceptive because it is widely present in tissue cells. Our results show that endogenous biotin is present in frozen tissue, and biotin is blocked by formalin-fixed paraffin. Endogenous biotin exposure caused by heating antigen repair, the intensity of endogenous biotin exposure varies from tissue to tissue, from weak positive ( + ) to strong positive ( +++ ), endogenous biotin The distribution form in the tissue, both scattered and diffuse, mainly exists in the cytoplasm in the form of granules. Endogenous biotin is widely present in epithelial-derived tissues, especially glandular epithelial tissues, and some non-epithelial tissues are also present. Endogenous biotin not only exists in human tissues but also in rat tissues. The exposure of endogenous biotin is related to the repair fluid. The increase in strength is: citric acid, EDTA , EGTA , endogenous exposure to thermal antigen repair The biotin can be blocked by egg white, and the non-biotin detection system Polymer two-step method ( EliVision , EnVision ) can avoid biotin interference.
7 , nuclear staining
Inadequate tissue treatment can cause nuclear staining, such as tissue immersion in xylene for too long (such as immersion from Friday to next Monday), soaking in buffer for too long, tissue drying, microwave repair fluid pH and Improper repair time or repair solution left too little, no tissue, etc. The solution is to work strictly in accordance with the operating rules.
6 , interstitial coloring
The coloring site is mainly in the interstitial, and there are many reasons for the interstitial staining. For example, the antibody and the protein in the tissue are colored by the interaction of the hydrophobic group of the protein to form a non-specific linkage. The step of blocking the serum before the primary antibody is to avoid non-specific Type of combination. In another example, immunoglobulins in serum often exude into the interstitial tissue, which easily binds to antibodies, causing interstitial staining, especially when lambda and kappa are stained. When the thyroid gland overflows into the interstitial tissue, interstitial staining also occurs in thyroglobulin staining. Interstitial staining can also occur if the antibody is impure or the antibody is contaminated. We have encountered impure CD20 antibodies, in addition to staining the B cells and staining the stroma.
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