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361
1. No signal
Antibody causes
1. Problems with the activity of primary and secondary antibodies, such as insufficient activity of the antibody reagents themselves, antibodies out of shelf life, repeated use of antibodies, contamination of antibodies, inappropriate concentration of antibodies used, etc.; can be done by selecting antibody reagents with quality assurance and activity verification and following the instructions at.
2. Primary and secondary antibodies problem one cannot recognize each other, for example, primary antibodies select mouse-derived antibodies and secondary antibodies select anti-rabbit antibodies or recognize antibodies from other species, resulting in secondary and primary antibodies not binding to each other.
Sample and reagent problems
3. Slices are too old and it is recommended to keep samples at 4°C.
4. The use of overly old samples may result in loss of staining signal. Different target proteins may have different weakening for antibody binding activity after storage for a period of time, and if sections need to be stored, it is recommended that they be stored at a constant temperature of 4°C.
5. Insufficient sensitivity of detection reagents, insufficient time, etc.
6. For some cases where there may be weak signal detection using only HRP-labeled secondary antibodies, higher-sensitivity secondary antibody-labeled antibodies can be used for detection of lowexpression targets.
7. Low or no antigen expression in the tissue section itself.
8. It is recommended that a positive control be set up in the experimental procedure to exclude the possibility that the experimental procedure, the improper use of reagents leading to negative results, and the possible existence of true negative samples at specific target sites such as phosphorylation.
Reasons for the experimental process
9. Incomplete antigen repairAntigen repair experimental steps can be used in a hot water bath, microwave oven or autoclave, you can also use protease for repair,according to the target to be detected to select the repair of the appropriate reagents (acid-base repair, etc.) to ensure the best conditions of the buffer system, it is generally recommended to use the microwave heating method for antigen repair or high-pressure conditions for antigen repair, to avoid the lack of antigen repair and reagents caused by the weak signal.
10. Too long fixation time Improper selection of fixation reagents using inappropriate concentration, etc., such as the use of paraformaldehyde, formaldehyde, should also avoid antigen damage caused by too long fixation time, etc.
11. Insufficient cellular permeation, resulting in failure toadequately color antibodies To allow full penetration of the reagents and antibodies throughout the sample, Triton is usually used. to ensure complete permeation of the slide, a concentration of 0.5-1% is recommended.
12. Insufficient antibody incubation time Primary or secondary antibodies correspond to significantly different incubation times at different incubation temperatures, generally overnight at 4°C to ensure adequate binding.
2. High background
1. Improper use of antibodies causes background coloring to be too dark Antibody working concentration is not diluted in accordance with the instructions, or the effect of target expression abundance is not mapped using concentration may lead to non-specific coloration of the background, primary antibody or secondary antibody incubation temperature and time is not appropriate to lead to elevated background coloration, the recommended incubation conditions are not 4 C overnight incubation.
2. Color development reagent color development time is too long, nottimely termination of color developmentColor development requires strict control of color development time under the microscope, not just a rough calculation of color development time.
3. Inadequate closure of slicesEnsure that the section is completely closed, before the incubation of the primary antibody, use a suitable closure solution for closure, the closure solution is generally selected from the same species as the source of the secondary antibody, such as the secondary antibodyis sheep anti-rabbit, the closure solution should be selected from sheep serum.
4. Consider increasing the number of dip washes and extending the dip time after antibody incubation to avoid insufficient number of washes or time resulting in too dark background coloration.
5. Avoid samples containing endogenous antigens Some background signals in the experimental procedure may come from the sample itself. For example, when using HRP detection system,l endogenous peroxidase in the sample will produce very strong background signal, so endogenous peroxidase should be eliminated in advance; if the target is some immunoglobulin, it may lead to false positive signal of secondary antibody, etc.
6. Mechanical damage or dry film, incomplete dewaxing resulting in background coloring problems When performing the dewaxing process, ensure that the slide is at the right temperature, put the solvent in quickly, and perform the dewaxingAvoid dry slides when performing antigen repair, primary antibody incubation, etc.
3. Presence of non-specific staining in tissue sectionsAntibody causes
1. Excessive antibody incubation time and high antibody concentration tend to increase background coloration. This can be controlled by shortening the incubation time of primary/secondary antibodies and diluting the antibodies. Appropriate antibody selection and incubation conditions are very important.
2. Use of monoclonal antibodies Although polyclonal antibodies have the advantages of abundant epitopes and high affinity, they can also suffer from phenomena such as non-specific binding, and the use of monoclonal antibodies can effectively avoid such causes of non-specific pooling.
3. For some samples where the secondary antibody is of the same species as the sample source, the secondary antibody may bind endogenous IgG, resulting in a high background
Reasons for the experimental process
4. The sample contains endogenous biotin, resulting in non-specific color development of the substrateThe endogenous peroxidase activity in the sample may produce an overly strong background signal.
5. Insufficient closureBefore incubation of the primary antibody, use a suitable blocking solution for closure. The blocking solution is generally selected from the same species as the source of the secondary antibody, such as the secondary antibody is sheep anti-rabbit, the blocking solution should be selected from sheep serum.
6. Excessive incubation time or high concentration of color developing reagent;Using the HRP color development system, DAB uses too high a concentration, or the background is too dark or non-specific due to inappropriate termination times.
7. Dry staining of specimens often occurs during staining, which tendsto enhance nonspecific staining.Throughout the experimental process, incubation conditions should be strictly controlled to avoid dry flakes caused by evaporation of reagents, processes, etc.
8. Adequate washing is critical for comparing low background and strong signals.Make sure to wash at least 3 times with PBS between steps to remove the effects of other reagents such as fixatives, hydrogen peroxide, etc.
4. Incomplete fixed tissue morphology
1. Inadequate or excessive fixationEnsure that the sample has been fixed long enough to allow the fixative to penetrate the sample. The fixation time may require some optimization. Fixation conditions are generally performed at 4°c.
2. Mechanical damageSamples are processed by moving tissue from one vial to another, or by adding or removing reagents, using a plastic Pasteur pipette with the tip removed (this prevents any damage to the embryo from the narrow end of the pipette). Avoid the use of forceps whenever possible.
5. Tissue section and exfoliation
Tissue desquamation during the staining process will not only waste precious tissue samples but also delay the experimental time, whichgenerally causes tissue desquamation mainly caused by the experimental process and materials used.
1. Slides containing polylysine are used for tissue sectioning, and slides with polarities and high binding activity are used to increase the stability of section binding.
2. Increase the baking time and adjust the appropriate temperature as appropriate. If there is a large-scale flaking phenomenon, it is often a problem in the experimental process, which can extend the baking time and increase the temperature of the flakes;
3. In addition to the above precautions, the PBS should be rinsed as gently as possible, avoiding direct rinsing against the sample, and it is recommended to choose drenching to reduce the physical impact caused by debonding.
4. Ce11 desquamation caused by a short period of sudden heat and coldin temperature when performing antigen repair repair.
5. Desquamation caused by buffer boiling when using antigen thermal repair and microwave repair.
6. Edge effects
1. Tissue edge is not firmly attached to the slide, and the thicknessof the tissue section is not appropriate Preparation of slides with strong adhesion, control of tissue section thickness, not thicker than 4 μm, and pre-treatment of tissues should avoid the selection of tissues with more necrosis to avoid sample-induced detachment and edge effects;

2. The reagent added dropwise to the slice does not adequately cover the tissue, resulting in a dry edge and thus an edge effect The reagent covers the tissue adequately and should extend beyond the edge of the tissue. When drawing circles with the histochemical pen, the appropriate distance from the sample should not be too close, generally controlled at about 3 mm.