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Location: Home > Information Center > FAQs > Pathology Detection-Immunohistochemistry (IHC)FAQs and So

Pathology Detection-Immunohistochemistry (IHC)FAQs and So

Date: 2016-05-30 Author: Leading Biology Click: 1420


Pathology Detection - Immunohistochemistry (IHC) FAQs and Solutions


Immunohistochemistry (IHC) is a technique that detects and localizes certain chemical substance in tissues and cells utilize specific binding reactions of antigen-antibodies. Technically, IHC experiments are not difficult, but there are many variable quantities in the experiment, making easy to encounter a variety of problems.


We listed frequently asked questions in IHC experiments and the corresponding solutions below.


Q1: Edge effect?

1)The edge of the tissues and the slides were not firmly adhered. The edge tissues were loosen and float in the liquid, and it is hard to clean the reagents under the tissues completely every time.


Solution: Prepare high-quality films (APES or poly-lysine) and cut off the thinnest tissue sections (no thicker than 4 um). The pre-treatment of the tissues should be standardized. Try to avoid using the tissues with much necrosis.


2)The reagents added on the slice didn’t cover the tissue fully. The reagents at the edge are easy to dry out first, which leads to a dark staining due to a higher concentration than that of the central tissues.


Solution: The reagents should cover the tissues fully and should be 2 mm beyond the edge of the tissues. In order to avoid the influence of oily reagents, the circle should be 3-4 mm away from the edge of the tissues when circling with a PAP pen.


Q2: What are the reasons for non-specific staining of tissue sections? How to solve?

1)A long time incubation and high concentration of antibodies are easy to deepen the background color. This can be controlled by shortening the incubation time of the primary/secondary antibodies and diluting the antibodies. This is the most important one.


2)It’s easy to stain non-specifically with polyclonal atibodies. Recommend to use monoclonal antibodies.


3)Endogenous peroxidases and biotin are highly contained in tissues such as liver and kidney (tissues with more hemocytes), and it is necessary to reduce background staining by extending inactivation time and increasing inactivating agent concentration.


4)It is necessary to enhance the blocking effect by extending the blocking time with animal immune serum sourcing from secondary antibodies and increasing the concentration appropriately during the non-specific component and the antibody binding.


5)DAB incubated for a long time or the concentration was too high.


6)Antibody remained with insufficient PBS washing results in enhanced staining. The washing after primary antibodies, secondary antibodies or SP incubation are particularly important.


7) Samples often dry out during staining, which tends to enhance non-specific staining.


Q3: What are the reasons for the negative results?

1)Incorrect selection of antibody concentration, quality and source. It’s wrong that the higher the antibody concentration, the more likely the positive result appears. There is pro- and post-zone phenomenon in antigen-antibody reaction, and the optimal concentration must be explored.


2)Incomplete antigen repaired. For formaldehyde-fixed tissues, sufficient antigenic repair must be used to open the epitope in order to bind with the antibody. Recommend to repair with microwave in high fire for 4 times and each time for 6 minutes. This is the best time in someone’s experiments. If not, it also can be repaired at high pressure.


3)Low antigen content in the tissue section itself.


4)The serum blocking time was too long.


5)DAB incubation time was too short.


6)The cells were incompletely penetrated, and the antibody didn’t fully enter the cell to react.


7) A positive control must be done to exclude method problems such as antibodies.


Q4: What are the reasons for strong background?

1)A high concentration of primary antibodies.


2)Adjust the DAB incubation time.


3)The serum blocking time is too short.


4) Increase the number of times of washing after incubation and extend the immersion time appropriately.


Q5: What are the reasons for wax slices dropped during the staining?

1)The baking time or the temperature was not enough.


2)Use a slide containing poly-lysine.


3)Some tissues are easy to fall off such as bone tissue. Do not rinse the PBS directly onto the tissue. Rinse to the top of the tissue and let it flow down to wash the tissue.


4)The temperature decreased suddenly during high temperature repair.


Q6: Why should I perform a 37°C degree rewarming after take the primary antibody out from 4°C?

1)Prevent the slices from dropping down.


2) Bind the antigen and antibody more stable. Usually, it is not needed. But it may be useful for weak expressed antigens. The molecular movement is different in 4°C and 37°C. The molecular collision probability and movement speed are lower in 4°C than 37°C. It combines faster in 37°C, but the sensitivity is also improved and easy to result in non-specific staining.


Q7: How to distinguish between specific and non-specific staining in the dark background?

Whole-slice coloring means the entire slice is stained light of dark. It is hard to distinguish the positive and negative tissues. The reasons are as follows:


1)Antibody concentration is too high: This is one of the most common reasons. The solution is to test the working concentration of new antibody before using. Individualize each antibody to find the ideal working concentration for your laboratory, even if it is a ready-to-use antibody. Do not stain simply according to the recommendations on the datasheet.


2) The antibody incubation time is too long or the temperature is too high: The solution is to follow the operating procedures strictly. Take a clock to avoid a long time incubation. The current popular two-step method (Polymer) is highly sensitive. The primary antibody incubation time is 30 minutes rather than conventional 1 hour. Therefore, it is necessary to adjust the time according to the results.


3) DAB deterioration and color development time is too long: DAB is better to use up after preparation. It should be filtered if there is sediment exist. The prepared DAB should not be stored for too long, because in the absence of enzymes, hydrogen peroxide will also release oxygen atoms to react with DAB to reduce the effectiveness. Store the remainder in the refrigerator for a few days is not desirable. The color development of DAB is better to be monitored under a microscope, and terminate the reaction immediately once the desired staining is achieved. However, it seems unrealistic when there are too many samples or dyeing with a machine, but at least some new or less-used antibodies should be monitored to avoid a long time color development.


4)Tissue dry out: The liquid is not replenished in time, the staining slice is too much, act too slow, forget to drop the liquid, and the drop liquid loss after the repair liquid overflows can cause the tissue dry out. Operate carefully and circle around the tissue with a DAKO pen or PAP Pen can effectively avoid liquid loss and improve the speed.


5)Immerse the slice in the buffer or repair solution for too long (more than 24 hours): The reason is not clear, but the phenomenon exists. Someone prefer to dewax the slices the day before, and add antibodies for immunohistochemical staining the next day. There is no significant influence if the container containing the slice and repairing solution is placed in a 4°C refrigerator overnight. If the container is placed at room temperature, especially in hot summer, the background coloration. So it should not be stored for too long.


6) Polyclonal antibodies with poor quality or go bad: Pay attention to the expiration date of the antibody. Using the expired antibody either does not develop color or background coloration. It is better to set up a positive control to compare with the used antibody when using a newly purchased antibody.


Q8: What are the reasons for the slice dropping down?

1)The low-quality of poly-lysine slides.


2)The tissue was not cut well. The problems of the microtome, for example, cut thick or uneven slices with an old machine or a bad slicing technique.


3)Not baked well, time is short, temperature is not enough, etc.


4)Shook the slice fiercely. Don’t shake or shake the slice gently. Absorb the water from the edge with toilet paper.


5)Repair problems: Repair the antigen with high pressure for too long or throw the slice into the 100°C repair solution. What’s more, it’s easy to release using EDTA than citric acid.


6)Be cautious once you see the tissue drifting up. Immerse in the PBS rather than flush.


7) Tissue problems. The more cancer tissue is necrotic, the easier it is to drop down.


Q9: Uneven coloration after DAB color development, for example, some slices were colored while some were not, or dyed light.

1)The thickness of the slices may not be the same. Some were thick while some were thin, which makes uneven coloration.


2)The slide didn’t shake after adding the coloring liquid, making inconsistent substrate concentration. It’s better to shake the slide back and forth after adding the coloring liquid to make a consistent concentration in all the areas.


3) If your slice is dyed dark in the middle and light near the edge, it is possible that your wax circle is too small, and the edge didn’t covered by the coloring liquid. It’s better to leave 0.5 cm between the outermost tissue piece and the edge of the coloring liquid.


Q10: Strong staining

1)The antibody titer was decreased because of a high antibody concentration or a long antibody incubation time (generally referred to as concentrated antibody).


2)The incubation temperature was too high.


3) The DAB color development time was too long or the DAB concentration was too high. The color development should not exceed 5-10 minutes according to the results observed under the microscope.


Q11: Non-specific background staining

1)Insufficient washing during operation.


2)The peroxidase contained in the tissue was not blocked. Prepare fresh 3% H2O2 to block or extend the incubation time.


3)The tissue contains endogenous biotin. Block again with normal non-immune animal serum.


4) Insufficient serum protein blocking. Extend the block time.


Q12: Light staining

1)The antibody concentration was too low or the incubation time was too short. Increase the antibody concentration and extend the incubation time to more than 60 minutes.


2)Replace the expired reagents in time.


3)The buffer was not dried before adding the reagents, which lead to a dilution. Dry the excess buffer before adding the reagents (Avoid drying out).


4)The room temperature was too low (below 15°C). If the room temperature is lower than 15 degrees, incubate in a 37°C incubator for 30-60 minutes (or 4°C overnight).


5) Excessive protein blocking. The blocking time should not exceed 10 minutes.


Q13: Negative staining

1)Incorrect operation steps. Do it again and set a positive control group.


2)No antigen in the tissue. Set a positive control group to test the result.


3) Wrong species connection between the primary and secondary antibody. Confirm the species carefully.


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