One. No Signal
1. The primary antibody does not match the secondary antibody.
Solution: Choose a secondary antibody that is specific to the species in which the primary antibody was raised. For example, if your primary antibody is from a rabbit, use a secondary antibody that targets rabbit IgG.
2. The amount of primary antibody bound to the target protein is insufficient.
Solution: Increase the concentration of both primary and secondary antibodies. Extend incubation time at 4°C (e.g., overnight) to improve binding efficiency.
3. The antibody may not be suitable for tissue immunohistochemistry, where proteins maintain their natural 3D structure.
Solution: First validate the antibody using reduced immunoglobulin samples (not denatured as in Western blot) to confirm its reactivity.
4. The primary/secondary antibody or amplification kit may have been inactivated due to improper storage, incorrect dilution, or repeated freeze-thaw cycles.
Solution: Always include a positive control to ensure the antibodies are functioning properly before proceeding with the experiment.
5. The tissue being studied does not express the target protein.
Solution: Use a recommended positive control provided by the antibody manufacturer to verify the system's functionality.
6. The target protein is expressed at low levels in the tissue.
Solution: Consider adding an amplification step to enhance signal detection.
7. The secondary antibody is not protected from light.
Solution: Store and handle secondary antibodies in dark conditions to prevent photodegradation.
8. Dewaxing is incomplete.
Solution: Increase dewaxing time and replace xylene with fresh solvent to ensure complete removal of paraffin.
9. Antigen epitopes may be masked during the fixation process.
Solution: Perform antigen retrieval techniques to expose hidden epitopes. Also, reduce fixation time to avoid over-fixation, which can lead to weak signals in the center of the sample.
10. The target protein is located in the nucleus, making it inaccessible to the antibody.
Solution: Add a permeabilization agent like Tween 20 or Triton X-100 to the wash and antibody dilution solutions. Adjust the concentration based on the thickness of the tissue section.
11. The PBS buffer is contaminated with bacteria that degrade phosphate groups on the target protein.
Solution: Add 0.01% sodium azide to the PBS solution or use sterilized PBS to prevent microbial contamination.
12. Exposure time during signal detection is too short.
Solution: Increase the exposure time during image capture to allow for better signal visualization.
Two. High Background
1. Incomplete or inadequate blocking.
Solution: Increase the blocking time or the serum concentration in the blocking solution. Replace the blocking reagent if necessary. We recommend blocking tissue sections with 10% normal serum for 1 hour, and cultured cells with 1–5% BSA for 30 minutes.
2. The primary antibody concentration is too high.
Solution: Reduce the concentration of the primary antibody to minimize non-specific binding.
3. Incubation temperature is too high.
Solution: Incubate the sections or cells at 4°C to reduce non-specific interactions.
4. Non-specific binding of the secondary antibody.
Solution: Include a secondary-only control to identify any non-specific staining caused by damaged or cross-reactive antibodies.
5. Residual fixative remains in the tissue.
Solution: Wash thoroughly between all steps using TBS or PBS to remove unbound fixatives.
6. Endogenous peroxidase activity is present.
Solution: Inhibit endogenous enzymes by adding an inhibitor such as 2 mM levamisole (for alkaline phosphatase) or 0.3–3% hydrogen peroxide (for peroxidase).
7. Overly strong fixation has altered the antigen recognition site.
Solution: Optimize antigen retrieval methods and reduce the incubation time with the antigen unmasking solution.
8. Excessive amplification occurs.
Solution: Shorten the amplification incubation time and dilute the amplification reagent if needed.
9. Too much substrate is used in enzyme-based detection systems.
Solution: Decrease the incubation time with the substrate to prevent overdevelopment.
10. Chromogen reacts with PBS in tissues or cells.
Solution: Wash the sections with TBS before and after substrate incubation to eliminate background reactions.
11. Penetrant treatment damages the cell membrane and removes membrane-bound proteins.
Solution: Avoid using penetrants unless absolutely necessary. If required, use them at minimal concentrations.
Three. Non-Specific Staining
1. The concentration of primary or secondary antibody is too high.
Solution: Lower the antibody concentration or shorten the incubation time. Monitor signal intensity in negative control cells to guide optimization.
2. Endogenous peroxidase activity is still active.
Solution: Continue using enzyme inhibitors such as hydrogen peroxide or levamisole to block unwanted enzymatic activity.
3. The tissue source is from the same species as the primary antibody (e.g., mouse primary on mouse tissue).
Solution: Select primary antibodies from a different species than the tissue being tested to avoid cross-reactivity with secondary antibodies.
4. The tissue or cells dry out during the procedure.
Solution: Keep the sections or cells in a humid environment throughout the procedure to prevent drying and preserve integrity.
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