One. No Signal
1. The primary antibody does not match the secondary antibody. Solution: Ensure that the secondary antibody is raised against the same species as the primary antibody. For example, if the primary antibody was produced in a rabbit, use a secondary antibody specifically targeting 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 the incubation time at 4°C, such as overnight, to allow better binding. 3. The antibody is not suitable for immunohistochemistry, where proteins maintain their native 3D structure. Solution: Verify the antibody’s suitability for IHC by testing it on a reduced sample (not a Western blot), or consult the manufacturer's recommendations. 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 use a positive control to confirm that your reagents are functional and working properly. 5. The tissue being studied does not express the target protein. Solution: Use a positive control recommended by the antibody supplier to validate the system. 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 exposed to light. Solution: Store secondary antibodies in the dark and avoid exposure to light during handling. 8. Dewaxing is incomplete. Solution: Extend the dewaxing time and replace the xylene with fresh solution to ensure proper removal of paraffin. 9. The fixation process may block the epitope. Solution: Perform antigen retrieval techniques to expose hidden epitopes. Reduce fixation time if necessary. Over-fixation can lead to weak central staining and strong edge staining. 10. The target protein is located in the nucleus, preventing antibody access. Solution: Add a permeabilizing agent like Tween 20 or Triton X-100 to the wash or antibody diluent. 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 antibody diluent or use sterile PBS to prevent bacterial growth. 12. The exposure time is too short. Solution: Increase the exposure time during imaging or signal acquisition to capture a stronger signal. Two. High Background 1. Incomplete or inadequate blocking. Solution: Increase the blocking time or the serum concentration in the blocking solution. For tissue sections, use 10% normal serum for 1 hour; for cultured cells, use 1–5% BSA for 30 minutes. 2. The primary antibody concentration is too high. Solution: Optimize the concentration of the primary antibody to reduce non-specific binding. 3. Incubation temperature is too high. Solution: Incubate samples at 4°C to minimize non-specific interactions. 4. Non-specific binding of the secondary antibody. Solution: Run a secondary-only control to check for non-specific binding and ensure the secondary antibody is functioning correctly. 5. Residual fixative remains on the tissue. Solution: Wash thoroughly between each step using TBS or PBS to remove any remaining fixative. 6. Endogenous peroxidase activity is present. Solution: Inhibit endogenous enzymes by using appropriate inhibitors such as 2 mM levamisole for alkaline phosphatase or 0.3–3% hydrogen peroxide for peroxidase. 7. Overly aggressive fixation alters antigen recognition. Solution: Try different antigen retrieval methods and reduce the time spent in the unmasking solution. 8. Excessive amplification leads to background. Solution: Shorten the amplification incubation time and dilute the amplification reagent as needed. 9. Too much substrate used in enzyme-based detection. Solution: Reduce the incubation time with the substrate to avoid overdevelopment. 10. Chromogen reacts with PBS in tissues or cells. Solution: Wash sections with TBS before and after substrate incubation to prevent unwanted reactions. 11. Permeabilization disrupts cell membranes and removes membrane-bound proteins. Solution: Avoid using permeabilizers unless absolutely necessary, especially when targeting membrane proteins. Three. Non-Specific Staining 1. Antibody concentration or incubation time is too high. Solution: Lower the antibody concentration or shorten the incubation time. Monitor the signal intensity in negative control cells. 2. Endogenous peroxidase activity is still present. Solution: Continue using enzyme inhibitors to block non-specific signals from endogenous enzymes. 3. The tissue source is from the same species as the primary antibody. Solution: Choose a primary antibody from a different species than the tissue being tested to avoid cross-reactivity. 4. The tissue or cell section dries out. Solution: Keep sections in a humid environment to prevent drying, which can cause nonspecific staining and loss of signal.Foshan Liqia Hardware Products Co., Ltd. , https://www.liqiamei.com