Q1: Edge effect?
1) The edge of the tissue and the slide are not firmly adhered, and the edge tissue is loosened and floated in the liquid. It is not easy to wash the reagent under the tissue every time. Solution: Prepare high-quality film (APES or polylysine), Cut as thin a tissue section as possible, 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 2mm beyond the edge of the tissue. When using a group stroke to draw a circle, in order to avoid the influence of oil, the circle should be 3-4mm from the edge of the tissue.
Q2: What are the reasons for non-specific staining of tissue sections? How to solve them?
1) The incubation time of the antibody is too long, and the antibody concentration is high, which tends to increase the background coloration. This can be controlled by shortening the incubation time of the primary/secondary antibody and diluting the antibody. This is the most important one.
2) Polyclonal antibodies against primary antibodies are prone to non-specific staining. It is recommended to try monoclonal antibodies.
3) Endogenous peroxidase and biotin are highly contained in tissues such as liver and kidney (hepatocytes containing many blood cells), and it is necessary to reduce background staining by prolonging the inactivation time and increasing the concentration of inactivating agent;
4) The non-specific component binds to the antibody, which needs to enhance the blocking effect by prolonging the blocking time of the immune serum of the secondary antibody source and increasing the concentration appropriately;
5) DAB incubation time is too long or the concentration is too high;
6) Insufficient PBS washing, residual antibody results in enhanced staining, and dip after primary, secondary or SP incubation is particularly important;
7) Dry sheets often appear during specimen staining, which tends to enhance non-specific staining.
Q3: What are the reasons for the negative results of immunohistochemical staining?
1) Antibody concentration and quality issues and antibody source selection errors. The higher the antibody concentration, the more likely the positive result is. The antigen-antibody reaction has a anterior and posterior band effect, and the optimal concentration must be explored.
2) Antigen retrieval is incomplete. For formaldehyde-fixed tissues, sufficient antigenic repair must be used to open the epitope to facilitate binding with the antibody. It is recommended to use microwave for 4 times*6min. Someone has done experiments, this is the best time and number of times. If not, it can be repaired at high pressure.
3) The tissue slice itself has a low antigen content;
4) Serum blocking time is too long.
5) DAB incubation time is too short.
6) The cells are incompletely permeable, and the antibody does not fully enter the cell to participate in the reaction.
7) To start immunohistochemistry, be sure to first make a positive photo, and exclude methods such as antibodies.
Q4: Why is the background strong?
1) Consider the high concentration of primary antibody;
2) then adjust the DAB incubation time;
3) Also consider whether the serum closure time is too short
4) Appropriately increase the number of times of immersion after incubation of the antibody and prolong the immersion time.
Q5: Wax slices appear to be stripped during the dyeing process?
1) The baking time is not enough, or the temperature is not enough, it can extend the baking time and increase the temperature of the baking sheet.
2) Use a slide containing polylysine, you can buy it or do it yourself
3) Some tissues are easy to fall off, such as bone tissue. When operating, do not rush the PBS directly onto the tissue, rush to the top of the tissue, let it flow down to wash the tissue.
4) When using high temperature repair, temperature quenching may also occur.
Q6: Why should I perform a 37 degree rewarming after the primary antibody is taken out at 4 degrees?
1) On the one hand, prevent the slices from being directly placed into the PBS easily released from 4 degrees;
2) On the other hand, the antigen-antibody binding is made more stable. Generally not needed, but may be useful for antigens with weak expression. At 4 and 37 degrees, the molecular motion is different. The former has a lower molecular collision probability and movement speed than the latter. The latter combines faster, but the sensitivity is also improved. Causes non-specific staining.
Q7: The background is too dark after section staining. How to distinguish between specific and non-specific staining?
Whole-slice coloration 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) Excessive antibody concentration: High concentration of primary antibody is one of the common causes. The solution is to test the working concentration 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, not simply 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 the delay caused by forgetting. The current 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. The prepared DAB should not be stored for too long, because in the absence of enzymes, hydrogen peroxide will also liberate oxygen atoms to react with DAB to reduce the effectiveness of DAB. The unused DAB is stored in the refrigerator for 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) Drying of the tissue: the liquid is not replenished in time after the overflow of the repair solution, too much stained slice, 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 PAP Pen to draw a circle 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 repair the day before, and add antibodies for immunohistochemical staining the next day. If the container containing the sectioning and repairing solution is placed in a 4oC 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) Polyclonal antibodies with poor resistance and poor quality: Pay attention to the expiration date of the antibody. The expired antibody either does 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.
Q8: What are the reasons for the release?
1) The problem of the quality of polylysine slides.
2) The tissue is not cut well. The problems of the microtome are, for example, the thick or uneven cut of the old machine, or the bad technique of the slicer.
3) Not baked, time is short, temperature is not enough.
4) When the operation is too fierce, the film suspected of peeling off is best not to rub or gently rub, use the toilet paper to slowly absorb water from the edge.
5) The problem of repair: the high pressure time is too long when the antigen is repaired, or the technique is not good when the 100 degree repair solution is put in, and the slamming sound is thrown in, so that it is easy to take off the piece. In addition, EDTA repair is easier than citric acid, but you can't use EDTA. You only have to start with another problem.
6) Be cautious once you see the organization drifting up. Use PBS as much as you can, don't rush. Basically, these aspects have been noticed, and improvements can be made as much as possible, and stripping can be reduced a lot.
7) Organizational problems, I use a lot of tissue cancer, the more cancer tissue is necrotic, the easier it is to take off.
Q9: After DAB color development, it is found that the slice coloration is uneven: such as a piece of coloring, one piece is not colored or dyed light?
1) The cut thickness of the film itself may be different. Cut a thick piece and a thin piece, which may cause different shades of color.
2) It may be that your coloring liquid substrate is not shaken after being added to the film, causing the local substrate concentration to be inconsistent. Therefore, after adding the coloring liquid, it is best to shake the film back and forth several times to make all the areas on the film. The substrate concentration is consistent.
3) If your film is dyed deep in the middle and shallow near the edge, it may be that your wax circle is too small, and the area covered by the coloring liquid is not too large and has an edge effect. The outermost tissue piece is preferably 0.5 cm from the outer edge of the color developing solution.
Q10: The dyeing is too strong
1) The antibody concentration is too high or the antibody incubation time is too long to reduce the antibody titer (generally referred to as concentrated antibody)
2) The incubation temperature is too high,
3) If the DAB color development time is too long or the DAB concentration is too high, the color development time should not exceed 5-10 minutes, which is subject to the observation under the microscope.
Q11: Non-specific background staining
1) Insufficient flushing during operation
2) The peroxidase contained in the tissue is not blocked, and can be re-configured with fresh 3% H2O2 for blocking, and the incubation time is prolonged.
3) Endogenous biotin contained in the tissue, which can be resealed by normal non-immune animal serum
4) Insufficient serum protein blocking can prolong serum protein blocking time
Q12: weak dyeing
1) The antibody concentration is too low, the incubation time is too short, and the antibody concentration can be increased. The incubation time should not be less than 60 minutes.
2) Reagents should be replaced in time for the effective use period
3) During the operation, the buffer is not drained when the reagent is added, so that the reagent is diluted. The excess buffer in the section can be drained before each step of adding the reagent (but the section is prevented from drying)
4) The room temperature is too low, below 15 °C If the room temperature is lower than 15 degrees, it should be incubated in a 37 ° C incubator for 30-60 minutes (or 4 ° C refrigerator overnight)
5) Protein is too closed, and the sealing time should not exceed 10 minutes.
Q13: Negative staining
1) The operation steps are incorrectly retested and a positive control is established.
2) Establish a positive pair of photos without antigen in the tissue to verify the results of the experiment
3) The connection between the primary antibody and the secondary antibody is carefully determined to ensure that the primary antibody and the secondary antibody are correct.
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