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Analysis and handling of IHC common problems
posted this at Jun 5, 2025 Updated at Oct 30, 2024
3
| Problem |
Reason |
Suggest |
| The result is too dark |
The concentration of the primary antibody is too high or the incubation time is too long |
Reduce primary antibody concentration or incubation time |
| The result is too dark |
The incubation temperature is too high |
Incubate at room temperature or 4 °C |
| The result is too dark |
The incubation time of the HRP-labeled secondary antibody is too long |
Reduce time |
| Non-specific staining in IHC results |
The paraffin section is not completely dewaxed |
Extend deparaffinization time |
| Non-specific staining in IHC results |
Insufficient rinsing during the procedure |
Increase the number and duration of washes |
| Non-specific staining in IHC results |
The tissue is rich in endogenous biotin and peroxidase |
Block with relevant reagent |
| Non-specific staining in IHC results |
Antigenic ectopia occurs |
|
| Non-specific staining in IHC results |
Insufficient protein blocking |
Increase the protein blocking time |
| Weak color development in IHC results |
The concentration of the primary antibody is too low or the incubation time is too short |
Increase the concentration of the primary antibody or extend the incubation time |
| Weak color development in IHC results |
The reagent has expired |
Confirm the shelf life of the reagent before the experiment |
| Weak color development in IHC results |
Excessive rinsing during the procedure |
Rinse appropriately |
| No staining in IHC results |
Expression of non-target antigens in the tissue |
|
| No staining in IHC results |
mismatch between the species of the primary antibody and the secondary antibody |
Verify the species origin of the primary antibody before the experiment |
| No staining in IHC results |
mismatch between the chromogen and the HRP system |
Verify the color development system before the experiment | |
For more product details, please visit Aladdin Scientific website.
https://www.aladdinsci.com/
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